Hibiscus cannabinus feruloyl-coa:monolignol transferase

ABSTRACT

The invention relates to isolated nucleic acids encoding a feruloyl-CoA:monolignol transferase and feruloyl-CoA:monolignol transferase enzymes. The isolated nucleic acids and/or the enzymes enable incorporation of monolignol ferulates into the lignin of plants, where such monolignol ferulates include, for example, p-coumaryl ferulate, coniferyl ferulate, and/or sinapyl ferulate. The invention also includes methods and plants that include nucleic acids encoding a feruloyl-CoA:monolignol transferase enzyme and/or feruloyl-CoA:monolignol transferase enzymes.

This application claims benefit of the priority filing date of U.S.Provisional Patent Application Ser. No. 61/544,063, filed Oct. 6, 2011,the contents of which are specifically incorporated herein in theirentirety.

This application is related to U.S. Patent Application Ser. No.61/366,977, filed Jul. 23, 2010, and PCT/US2011/044981, filed Jul. 22,2011, the contents of both of which are specifically incorporated hereinby reference in their entireties. This application is also related topublished U.S. patent application Ser. No. 12/830,905, filed Jul. 6,2010 and to U.S. Patent Application Ser. No. 61/213,706, filed Jul. 6,2009, the contents of both of which are specifically incorporated hereinby reference in their entireties.

This invention was made with government support from Grant No.DE-FC02-07ER64494 awarded by the U.S. Department of Energy, Office ofBiological and the Environmental Research (BER) Office of Science. Thegovernment has certain rights in the invention.

This invention was made as a result of activities undertaken within thescope of a Joint Research Agreement between Michigan State Universityand Wisconsin Alumni Research Foundation.

BACKGROUND OF THE INVENTION

Lignin is an important cell wall component that provides structuralsupport to plants and is needed for plant vascular tissue function. Itis one of the most abundant organic polymers on Earth, constitutingabout 30% of non-fossil organic carbon and from a quarter to a third ofthe dry mass of wood. Because the chemical structure of lignin isdifficult to degrade by chemical and enzymatic means, lignin makes thetask of producing paper and biofuels from plant cell walls difficult.

SUMMARY OF THE INVENTION

The invention relates to the identification and isolation of newacyltransferase nucleic acids and polypeptides. The acyltransferaseenzyme is a Hibiscus cannabinus (Kenaf) feruloyl-CoA:monolignoltransferase (FMT, also called a monolignol ferulate transferase) thatproduces monolignol ferulates, which can be used for making plants thatcontain a readily cleavable lignin. Use of the feruloyl-CoA:monolignoltransferase nucleic acids and/or polypeptides in plants can simplify theprocesses used for making biofuels and paper from those plants becausethese plants have lignin that is more readily removed by chemicaltreatment or pretreatment. Other cloned or isolated enzymes with thesebeneficial properties are not currently available.

One aspect of the invention is an isolated nucleic acid encoding aHibiscus cannabinus (Kenaf) feruloyl-CoA:monolignol transferase, whereinthe nucleic acid can selectively hybridize to a DNA with a SEQ ID NO:8sequence. For example, the nucleic acid can selectively hybridize to aDNA with a SEQ ID NO:8 sequence under stringent hybridizationconditions. In some embodiments, the stringent hybridization conditionscomprise a wash in 0.1×SSC, 0.1% SDS at 65° C. Such an isolated nucleicacid can have at least about 79%, at least about 80%, or at least about90%, or at least 95% sequence identity with SEQ ID NO:8. In someembodiments, the isolated nucleic acid with the SEQ ID NO:8 sequenceencodes a Hibiscus cannabinus (Kenaf) feruloyl-CoA:monolignoltransferase.

Another aspect of the invention is an isolated nucleic acid encoding aHibiscus cannabinus (Kenaf) feruloyl-CoA:monolignol transferasepolypeptide with a SEQ ID NO:9 or SEQ ID NO:16 sequence.

Such feruloyl-CoA:monolignol transferases can catalyze the synthesis ofmonolignol ferulate(s) from monolignol(s) and feruloyl-CoA. For example,the monolignol can be coniferyl alcohol, p-coumaryl alcohol, sinapylalcohol or a combination thereof, and the feruloyl-CoA:monolignoltransferase can, for example, synthesize coniferyl ferulate, p-coumarylferulate, sinapyl ferulate or a combination thereof.

As described in more detail herein, the feruloyl-CoA:monolignoltransferase nucleic acids and polypeptides produce monolignol ferulates.Unlike most plant lignins, lignin that contains monolignol ferulates isreadily cleavable.

In some embodiments, the feruloyl-CoA:monolignol transferase nucleicacid encodes a feruloyl-CoA:monolignol transferase polypeptide with aSEQ ID NO:9 or SEQ ID NO:16 sequence. In other embodiments, the nucleicacids can, for example, encode a feruloyl-CoA:monolignol transferasethat can catalyze the synthesis of monolignol ferulate(s) from amonolignol(s) and feruloyl-CoA with at least about 50%, of the activityof a feruloyl-CoA:monolignol transferase with the SEQ ID NO:9 or SEQ IDNO:16.

Another aspect of the invention is a transgenic plant cell comprising anisolated nucleic acid encoding a feruloyl-CoA:monolignol transferase.The nucleic acid can include any of the feruloyl-CoA:monolignoltransferase nucleic acids described herein. For example, the nucleicacid can include a nucleic acid segment that can selectively hybridizeto a DNA with a SEQ ID NO:8 sequence, or a nucleic acid that encodes aSEQ ID NO:9 or 16 amino acid sequence, or a nucleic acid that encodes aferuloyl-CoA:monolignol transferase that can catalyze the synthesis ofmonolignol ferulate(s) from a monolignol(s) and feruloyl-CoA with atleast about 50%, of the activity of a feruloyl-CoA:monolignoltransferase with the SEQ ID NO:9 or SEQ ID NO:16.

Another aspect of the invention is an expression cassette comprising oneof the feruloyl-CoA:monolignol transferase nucleic acids describedherein that is operably linked to a promoter functional in a host cell.Such a nucleic acid can include a nucleic acid segment that canselectively hybridize to a DNA with a SEQ ID NO:8 sequence, or a nucleicacid that encodes a SEQ ID NO:9 or 16 amino acid sequence, or a nucleicacid that encodes a feruloyl-CoA:monolignol transferase that cancatalyze the synthesis of monolignol ferulate(s) from a monolignol(s)and feruloyl-CoA with at least about 50%, of the activity of aferuloyl-CoA:monolignol transferase with the SEQ ID NO:9 or SEQ IDNO:16. The expression cassette can further comprise a selectable markergene. In some embodiments, the expression cassette further comprisesplasmid DNA. For example, the expression cassette can be within anexpression vector. Promoters that can be used within such expressioncassettes include promoters functional during plant development orgrowth.

Another aspect of the invention is a plant cell that includes anexpression cassette comprising one of the feruloyl-CoA:monolignoltransferase nucleic acids described herein that is operably linked to apromoter functional in a host cell. Such a nucleic acid can include anucleic acid segment that can selectively hybridize to a DNA with a SEQID NO:8 sequence, or a nucleic acid that encodes a SEQ ID NO:9 or 16amino acid sequence, or a nucleic acid that encodes aferuloyl-CoA:monolignol transferase that can catalyze the synthesis ofmonolignol ferulate(s) from a monolignol(s) and feruloyl-CoA with atleast about 50%, of the activity of a feruloyl-CoA:monolignoltransferase with the SEQ ID NO:9 or SEQ ID NO:16. The plant cell can bea monocot cell. The plant cell can also be a gymnosperm cell. Forexample, the plant cell can be a maize, grass or softwood cell. In someembodiments, the plant cell is a dicot cell. For example, the plant cellcan be a hardwood cell.

Another aspect of the invention is a plant that includes an expressioncassette comprising one of the feruloyl-CoA:monolignol transferasenucleic acids described herein that is operably linked to a promoterfunctional in a host cell. Such a plant can be a monocot. Such a nucleicacid can include a nucleic acid segment that can selectively hybridizeto a DNA with a SEQ ID NO:8 sequence, or a nucleic acid that encodes aSEQ ID NO:9 or 16 amino acid sequence, or a nucleic acid that encodes aferuloyl-CoA:monolignol transferase that can catalyze the synthesis ofmonolignol ferulate(s) from a monolignol(s) and feruloyl-CoA with atleast about 50%, of the activity of a feruloyl-CoA:monolignoltransferase with the SEQ ID NO:9 or SEQ ID NO:16. The plant can also bea gymnosperm. For example, the plant can be a maize, grass or softwoodplant. In some embodiments, the plant is a dicot plant. For example, theplant can be a hardwood plant.

Another aspect of the invention is a method for incorporating monolignolferulates into lignin of a plant that includes:

-   -   a) stably transforming plant cells with the expression cassette        comprising one of the feruloyl-CoA:monolignol transferase        nucleic acids described herein to generate transformed plant        cells;    -   b) regenerating the transformed plant cells into at least one        transgenic plant, wherein feruloyl-CoA:monolignol transferase is        expressed in at least one transgenic plant in an amount        sufficient to incorporate monolignol ferulates into the lignin        of the transgenic plant.

For example, such a nucleic acid can be a nucleic acid that canselectively hybridize to a DNA with a SEQ ID NO:8 sequence, or a nucleicacid that encodes a SEQ ID NO:9 or 16 amino acid sequence, or a nucleicacid that encodes a feruloyl-CoA:monolignol transferase that cancatalyze the synthesis of monolignol ferulate(s) from a monolignol(s)and feruloyl-CoA with at least about 50%, of the activity of aferuloyl-CoA:monolignol transferase with the SEQ ID NO:9 or SEQ IDNO:16. The method can be used to generate a transgenic plant that isfertile. The method can further include recovering transgenic seeds fromthe transgenic plant, wherein the transgenic seeds include the nucleicacid encoding a feruloyl-CoA:monolignol transferase. The plantcontaining monolignol ferulates within its lignin can be a monocot. Theplant can also be a gymnosperm. For example, the plant can be a maize,grass or softwood plant. In some embodiments, the plant is a dicotplant. For example, the plant can also be a hardwood plant. Such amethod can further include stably transforming the plant cell(s) or theplant with at least one selectable marker gene. The selectable markercan be linked or associated with the expression cassette.

In some embodiments, the lignin in the plant that has the nucleic acidencoding a feruloyl-CoA:monolignol transferase can include at least 1%monolignol ferulate. In other embodiments, the lignin in the plant caninclude at least 5% monolignol ferulate, or at least 10% monolignolferulate, or at least 20% monolignol ferulate, or at least 25%monolignol ferulate. In further embodiments, the lignin in the plantincludes about 1-30% monolignol ferulate, or about 2-30% monolignolferulate.

The method for incorporating monolignol ferulates into lignin of a plantcan also include breeding the fertile transgenic plant to yield aprogeny plant, where the progeny plant has an increase in the percentageof monolignol ferulates in the lignin of the progeny plant relative tothe corresponding untransformed plant.

Another aspect of the invention is a lignin isolated from the transgenicplant comprising any of the feruloyl-CoA:monolignol transferase isolatednucleic acids described herein. Another aspect of the invention is awoody material isolated from the transgenic plant comprising any of theferuloyl-CoA:monolignol transferase isolated nucleic acids describedherein. The lignin or woody tissue can include any of the nucleic acidsdescribed herein that encode a feruloyl-CoA:monolignol transferase. Inother embodiments, the lignin or woody tissue can include any of theferuloyl-CoA:monolignol transferase amino acid or polypeptide sequencesdescribed herein.

Another aspect of the invention is a method of making a product from atransgenic plant comprising: (a) providing a transgenic plant thatincludes one of the isolated nucleic acids described herein that encodesa feruloyl-CoA:monolignol transferase; and (b) processing the transgenicplant's tissues under conditions sufficient to digest to the lignin; tothereby generate the product from the transgenic plant, wherein thetransgenic plant's tissues comprise lignin having an increased percentof monolignol ferulates relative to a corresponding untransformed plant.Such a corresponding untransformed plant is typically a plant of thesame species, strain and/or accession as the transformed plant. Theconditions sufficient to digest to the lignin can include conditionssufficient to cleave ester bonds within monolignol ferulate-containinglignin. In some embodiments, the conditions sufficient to digest to thelignin include mildly alkaline conditions. In some embodiments, theconditions sufficient to digest to the lignin include contacting thetransgenic plant's tissues with ammonia for a time and a temperaturesufficient to cleave ester bonds within monolignol ferulate-containinglignin. In some embodiments, the conditions sufficient to digest to thelignin would not cleave substantially any of the ether and carbon-carbonbonds in lignin from a corresponding plant that does not contain theisolated nucleic acid encoding the feruloyl-CoA:monolignol transferase.

Therefore, the invention embraces nucleic acids encoding aferuloyl-CoA:monolignol transferase enzymes, feruloyl-CoA:monolignoltransferase enzymes, as well as expression cassettes, plant cells andplants that have or encode such nucleic acids and enzymes, and methodsof making and using such nucleic acids, polypeptides, expressioncassettes, cells and plants.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A1, 1A2, 1B1 and 1B2 illustrate structural models for some typesof lignin polymers. FIGS. 1A1 and 1A2 show examples of lignin structureswith 25 units that may be found in a softwood (spruce). FIGS. 1B1 and1B2 show examples of lignin structures with 20 units that may be presentin a hardwood (poplar). [Ralph, J., Brunow, G., and Boerjan, W. (2007)Lignins. In: Rose, F., and Osborne, K. (eds). Encyclopedia of LifeSciences, DOI: 10.1002/9780470015902.a0020104, John Wiley & Sons, Ltd.,Chichester, UK]. The softwood lignin is generally more branched andcontains a lower proportion of β-ether units. Note that each of thesestructures represents only one of billions of possible isomers [Ralph,J., Lundquist, K., Brunow, G., Lu, F., Kim, H., Schatz, P. F., Marita,J. M., Hatfield, R. D., Ralph, S. A., Christensen, J. H., and Boerjan,W. Lignins: natural polymers from oxidative coupling of4-hydroxyphenylpropanoids. (2004) Phytochem. Revs. 3(1), 29-60]. Thus,these structures are merely illustrative of some of the linkage typesthat may be present different lignins. An “S” within a ring indicates asyringyl unit while a “G” within a unit indicates a guaiacyl unit.

FIG. 2A-2B show HPLC traces of assay mixtures generated to test forferuloyl-CoA:monolignol transferase activity using coniferyl alcohol andferuloyl-CoA as substrates. The UV 340 trace is the dashed line whilethe UV 280 trace is the solid line. FIG. 2A is a no enzyme control assaywhile FIG. 2B shows the HPLC-separated assay results when theferuloyl-CoA:monolignol transferase enzyme from Angelica sinensis ispresent in the assay mixture. The peaks are numbered to distinguish theseparated components of the assay as follows: 1) coniferyl alcohol (atabout 4.4 min); 2) feruloyl-CoA (at about 5.4 min); 3) ferulic acid(about 6.0 min); and 4) coniferyl ferulate (at about 9.8 min)

FIG. 3A-3B illustrate the NMR identification of coniferyl ferulate(CAFA). FIG. 3A shows the assigned proton NMR spectrum of the productisolated from a reaction of coniferyl alcohol and feruloyl-CoA using theferuloyl-CoA:monolignol transferase from Angelica sinensis. FIG. 3B is a2D ¹H-¹³C correlation (HSQC) spectrum of the same produced coniferylferulate, further authenticating the product; the tabulated ¹³C NMR dataare from the 1D ¹³C NMR spectrum with the quaternary (non-protonated)carbons assigned by long-range ¹H-¹³C correlation (HMBC) spectra (notshown). These spectra (and proton and carbon data) match those fromauthentic (synthesized) coniferyl ferulate.

FIG. 4A-4B shows HPLC separation of assay components where the assay wasfor feruloyl-CoA:monolignol transferase (FMT) activity usingferuloyl-CoA and p-coumaryl alcohol as substrates. The UV 340 trace isthe dashed line while the UV 280 trace is the solid line. FIG. 4A showsthe results of a no-enzyme control assay while FIG. 4B shows the resultsof the assay with the feruloyl-CoA:monolignol transferase from Angelicasinensis. The peaks are numbered to distinguish the separated componentsof the assay as follows: 1)_(p)-coumaryl alcohol (at about 3.5 min), 2)feruloyl-CoA (at about 5.5 min), and 3) p-coumaryl ferulate (at about9.0 min)

FIG. 5A-5B shows HPLC separation of assay components where the assay wasfor feruloyl-CoA:monolignol transferase (FMT) activity using sinapylalcohol and feruloyl-CoA as substrates. The UV 340 nm trace is thedashed line while the UV 280 nm trace is the solid line. FIG. 5A showsthe results of a no-enzyme control assay while FIG. 5B shows the resultsof the assay with the feruloyl-CoA:monolignol transferase from Angelicasinensis. The peaks are numbered to distinguish the separated componentsof the assay as follows: 1) sinapyl alcohol (at about 4.4 min); 2)feruloyl-CoA (at about 5.5 min); and 3) sinapyl ferulate (at about 9.4min)

FIG. 6A-6B shows HPLC separation of assay components where the assay wasfor feruloyl-CoA:monolignol transferase (FMT) activity using coniferylalcohol and p-coumaroyl-CoA as substrates. The UV 340 nm trace is thedashed line while the UV 280 nm trace is the solid line. FIG. 6A showsthe results of a no-enzyme control assay while FIG. 6B shows the resultsof the assay with the feruloyl-CoA:monolignol transferase from Angelicasinensis. The peaks are numbered to distinguish the separated componentsof the assay as follows: 1) coniferyl alcohol and p-coumaroyl-CoA (atabout 4.4 min), the overlapping peaks cause a slight UV 280 asymmetrydue to the coniferyl alcohol elution only slightly before thep-coumaroyl-CoA; and 3) coniferyl p-coumarate (at about 9.4 min)

FIG. 7A-7B shows HPLC separation of assay components where the assay wasfor feruloyl-CoA:monolignol transferase (FMT) activity usingcaffeoyl-CoA and coniferyl alcohol as substrates. The UV 340 nm trace isthe dashed line while the UV 280 nm trace is the solid line. FIG. 6Ashows the results of a no-enzyme control assay while FIG. 6B shows theresults of the assay with the feruloyl-CoA:monolignol transferase fromAngelica sinensis. The peaks are numbered to distinguish the separatedcomponents of the assay as follows: 1) coniferyl alcohol (at about 4.4min); and 2) caffeoyl-CoA (at about 2.4 min)

FIG. 8 illustrates SDS-PAGE analysis of size exclusion chromatographyfractions from IMAC-purified feruloyl-CoA:monolignol transferase. Theterm UF is an abbreviations for unfractionated purifiedferuloyl-CoA:monolignol transferase. The numbers 19 through 26 representSuperdex75 gel filtration fractions. The symbol (−) identifies fractionswith no feruloyl-CoA:monolignol transferase activity while the symbols(+), (++) and (+++) mark fractions with progressively increasedactivity.

FIG. 9 illustrates the synthetic scheme used to prepare authenticconiferyl ferulate, employing (i) acetic anhydride, pyridine; (ii)thionyl chloride; (iii) borane/tert-butylamine; (iv) triethylamine,dimethylaminopyridine; and (v) pyrrolidine.

FIG. 10 illustrates that transgenic Poplar tree leaves express anenzymatically active Angelica sinensis feruloyl-CoA:monolignoltransferase. The Poplar trees were genetically modified using standardprocedures to incorporate the Angelica sinensis FMT nucleic acidsdescribed herein. FIG. 10A illustrates GFP-trap Mag enrichment anddetection of FMT expression in the leaves of transgenic poplar treesthat express FMT that has been N-terminally tagged with YellowFluorescent Protein (YFP-FMT). A western blot is shown ofelectrophoretically separated fractions obtained after GFPtrap(Chromotek) enrichment of YFP-FMT from the leaves of the transgenicpoplar trees that express YFP-FMT. The FMT9 and FMT13 lanes containextracts from two different genetically modified Poplar trees. FMTexpression was detected using anti-GFP antibodies (Abcam). FIG. 10Billustrates the results obtained from a poplar leaf extract FMT enzymeassay. UPLC traces are of control and transgenic Poplar leaf extracts,where the transgenic Poplar trees express the YFP-FMT from Angelicasinensis. The absorbance of the substrates coniferyl alcohol (1) andferuloyl-CoA (2) are shown along with the FMT product, coniferylferulate (3), was detected at 280 nm (solid line) and 340 nm (dottedline). The top panel shows results obtained for wild-type Poplar leafextracts (containing no Angelica sinensis FMT nucleic acids) while thebottom panel shows results obtained from extracts of transgenic poplarleaves that express the Angelica sinensis FMT. Coniferyl ferulate (3)was detected only with the leaf extract from YFP-FMT Poplar.

FIG. 11 illustrates that transgenic Arabidopsis express an enzymaticallyactive Angelica sinensis feruloyl-CoA:monolignol transferase. FMTexpression is demonstrated by Reverse Transcriptase PCR in Arabidopsisleaf. FMT enzymatic activity is demonstrated within the Arabidopsisstem. FIG. 11A illustrates the products of Reverse Transcriptase PCRthat were amplified from Arabidopsis leaves transformed with emptyvector or with a vector expressing the FMT transcript, when reversetranscriptase is added (+RT) or not added (−RT) to the PCR reactionmixture. A PCR product of the expected size for FMT (1326 base pairs) isvisible only in the reaction containing total RNA from Arabidopsistransformed with the Angelica sinensis FMT when the reversetranscriptase is present. FIG. 11B provides representative UPLC tracesshowing FMT activity in ground stems from Arabidopsis transformed withthe FMT from Angelica sinensis, when the FMT enzyme assay is employed(bottom panel). The absorbance for each of the substrates, coniferylalcohol (1) and feruloyl-CoA (2) and for the product, coniferyl ferulate(3), was measured at 280 nm (solid line) and 340 nm (dotted line).Control reactions were conducted with stems expressing empty vector (toppanel). Coniferyl ferulate (3) is detected only when protein from thetransformed Arabidopsis-FMT stems was added.

FIGS. 12A and 12B illustrate the expression, purification and enzymeactivity for FMT from Hibiscus cannabinus. FIG. 12A illustrates Hibiscuscannabinus FMT expression in E. coli BL21 cells (Invitrogen). TheHibiscus cannabinus FMT was expressed with an N-terminal 6×His tag inthe pDEST17 vector (Invitrogen) and the soluble protein (˜50 kDa) waspurified over a Ni²⁺ column using an AKTA purifier (GE Healthcare).Fractions containing purified protein (fractions 29 and 30) were assayedfor FMT activity. FIG. 12B shows the products of an FMT enzyme assayafter UPLC separation and detection by absorbance at 280 nm (solid line)and 340 nm (dotted line) for the substrates coniferyl alcohol (1) andferuloyl-CoA (2). A control reaction with no enzyme is shown at the top.The reaction containing the Hibiscus cannabinus FMT enzyme is shown inthe bottom panel. The production of coniferyl ferulate (3) is visibleonly when the Hibiscus cannabinus FMT enzyme is present in the assay(bottom panel). The product and substrate peaks were identified bycomparison to synthetic standards.

FIG. 13 shows an alignment of the Hibiscus cannabinus (lower sequence,SEQ ID NO:16) and Angelica sinensis (upper sequence, SEQ ID NO:17)feruloyl-CoA:monolignol transferase sequences. As illustrated, theHibiscus cannabinus and Angelica sinensis feruloyl-CoA:monolignoltransferases share only about 23% sequence identity. When similar aminoacid substitutions are considered, the Hibiscus cannabinus and Angelicasinensis feruloyl-CoA:monolignol transferases share only about 41%sequence similarity.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting. The following ispresented by way of illustration and does not limit the scope of theinvention.

DETAILED DESCRIPTION

The invention provides nucleic acids and methods useful for alteringlignin structure and/or the lignin content in plants. Plants with suchaltered lignin structure/content are more easily and economicallyprocessed into useful products such as biofuels and paper.

Acyl-CoA Dependent Acyltransferases

Plant acyl-CoA dependent acyltransferases constitute a large butspecific protein superfamily, named BAHD. Members of this family take anactivated carboxylic acid (i.e., a CoA thioester form of the acid) as anacyl donor and either an alcohol or, more rarely, a primary amine, as anacyl acceptor and catalyze the formation of an ester or an amide bond,respectively. The acyl donors and acyl acceptors that act as substratesby BAHD acyltransferases are quite diverse, and different BAHD familymembers exhibit a range of substrate specificities.

The invention relates to a new type of BAHD acyltransferase nucleicacids and enzymes that enable the production of transgenic plants withaltered lignin. The BAHD nucleic acids can be used in the expressioncassettes, expression vectors, transgenic plant cells, transgenic plantsand transgenic seeds as described herein. The BAHD nucleic acids andencoded proteins are isolated or heterologous nucleic acids or proteins.The term “isolated” when used in conjunction with a nucleic acid orpolypeptide, refers to a nucleic acid segment or polypeptide that ispresent in a form or setting that is different from that in which it isfound in nature. For example, an isolated nucleic acid or an isolatedpolypeptide is identified and separated from at least one contaminantnucleic acid or polypeptide with which it is ordinarily associated inits natural state. In contrast, native nucleic acids, such as DNA, RNAand polypeptides are found in the state they exist in nature. The term“heterologous” when used in reference to a nucleic acid refers to anucleic acid segment that has been manipulated in some way. For example,a heterologous nucleic acid includes a nucleic acid segment from onespecies that has been introduced into another species. A heterologousnucleic acid also includes a nucleic acid segment that is native to anorganism that has been altered in some way (e.g., mutated, multiplecopies are added, the heterologous nucleic acid is linked to anon-native promoter or enhancer sequence, etc.). Heterologous nucleicacids can include plant nucleic acid segments such as cDNA forms of aplant gene where the cDNA sequences are expressed in a sense directionto produce mRNA. In some embodiments, heterologous nucleic acid can bedistinguished from endogenous plant genes in that the heterologousnucleic acid segments are joined to nucleotide sequences comprisingregulatory elements such as promoters that are not found naturallyassociated with the endogenous gene in its natural chromosome. In someembodiments, heterologous nucleic acid can be distinguished fromendogenous plant genes in that the heterologous nucleic acid segmentsexpress the encoded protein (or portion of a protein) in parts of theplant where the protein (or portion thereof) is not normally expressed.

The acyltransferases described herein are feruloyl-CoA:monolignoltransferases that synthesize monolignol ferulates from any of threemonolignols (p-coumaryl, coniferyl and sinapyl alcohols). For example,the feruloyl-CoA:monolignol transferases described herein can synthesizeconiferyl ferulate from coniferyl alcohol and feruloyl-CoA, as shownbelow.

The feruloyl-CoA:monolignol transferases enable production of plantswith lignin that is readily cleaved and/or removed, for example, becausethe lignin in these plants contains monolignol ferulates such asconiferyl ferulate (CAFA).

The terms “feruloyl-CoA:monolignol transferase(s)” and “monolignolferulate transferase(s)” are used interchangeably herein.

Nucleic acids encoding the feruloyl-CoA:monolignol transferases that areuseful for making coniferyl ferulate (and other monolignol ferulates)were isolated from the roots of Angelica sinensis as clone Dq155pdestl7. The coding region of the Angelica sinensis clone Dq155 pdestl7has the following nucleic acid sequence (SEQ ID NO:1).

1 ATGACGATCA TGGAGGTTCA AGTTGTATCT AAGAAGATGG 41TAAAGCCATC AGTTCCGACT CCTGACCACC ACAAGACTTG 81CAAATTGACG GCATTCGATC AGATTGCTCC TCCGGATCAA 121GTTCCCATTA TTTACTTCTA CAACAGCAGC AACATCCACA 161ATATTCGCGA GCAATTGGTA AAATCCTTGT CCGAAACTCT 201AACCAAGTTT TATCCATTAG CTGGAAGATT TGTTCAAGAT 241GGTTTCTATG TCGATTGTAA TGATGAAGGG GTCTTGTACG 281TAGAAGCTGA AGTTAACATT CCGCTAAACG AATTCATCGG 321ACAAGCAAAG AAAAATATAC AACTTATCAA TGATCTTGTT 361CCGAAAAAAA ACTTCAAGGA TATTCATTCA TATGAAAATC 401CAATAGTGGG ATTACAGATG AGTTATTTCA AGTGTGGTGG 441ACTTGCTATT TGCATGTATC TTTCGCATGT TGTAGCTGAT 481GGATATACAG CAGCAGCATT CACTAAAGAG TGGTCTAACA 521CAACCAATGG CATCATCAAT GGCGATCAAC TAGTTTCTTC 561TTCTCCGATT AACTTCGAAT TGGCAACTCT AGTCCCAGCT 601AGAGATTTAT CGACGGTGAT CAAGCCAGCC GTGATGCCAC 641CATCAAAGAT CAAGGAAACC AAGGTTGTCA CAAGGAGGTT 681TCTGTTCGAT GAAAATGCGA TATCAGCTTT CAAAGACCAT 721GTCATCAAAT CCGAAAGCGT TAACCGGCCT ACACGGGTGG 761AAGTTGTGAC ATCTGTGTTA TGGAAGGCTC TGATCAACCA 801GTCTAAGCTT CCAAGTTCTA CACTATATTT TCACCTCAAC 841TTTAGAGGGA AAACAGGCAT CAACACCCCA CCGCTAGATA 881ATCATTTTTC GCTTTGCGGA AACTTTTACA CTCAGGTTCC 921TACAAGGTTC AGGGGGGGAA ATCAAACAAA ACAGGATTTG 961GAATTGCATG AATTGGTCAA GTTGTTGAGA GGAAAGTTGC 1001GTAACACTCT GAAGAATTGC TCCGAAATTA ACACTGCCGA 1041TGGGCTGTTC CTGGAAGCAG CTAGTAATTT CAATATTATA 1081CAGGAAGATT TGGAGGACGA ACAAGTGGAT GTTCGGATTT 1121TTACAACGTT GTGTAGGATG CCTTTGTATG AAACTGAGTT 1161TGGGTGGGGA AAACCAGAAT GGGTTACCAT TCCAGAGATG 1201CATTTGGAGA TAGTGTTTCT TTTGGACACT AAATGTGGGA 1241CTGGTATTGA GGCATTAGTG AGCATGGATG AAGCAGATAT 1281GCTTCAGTTT GAACTTGATC CCACCATCTC TGCTTTCGCT 1321 TCCTAGThe SEQ ID NO:1 nucleic acid encodes an Angelica sinensis clone Dq155pdest17 feruloyl-CoA:monolignol transferase enzyme with the followingamino acid sequence (SEQ ID NO:2).

1 MTIMEVQVVS KKMVKPSVPT PDHHKTCKLT AFDQIAPPDQ 41VPIIYFYNSS NIHNIREQLV KSLSETLTKF YPLAGRFVQD 81GFYVDCNDEG VLYVEAEVNI PLNEFIGQAK KNIQLINDLV 121PKKNFKDIHS YENPIVGLQM SYFKCGGLAI CMYLSHVVAD 161GYTAAAFTKE WSNTTNGIIN GDQLVSSSPI NFELATLVPA 201RDLSTVIKPA VMPPSKIKET KVVTRRFLFD ENAISAFKDH 241VIKSESVNRP TRVEVVTSVL WKALINQSKL PSSTLYFHLN 281FRGKTGINTP PLDNHFSLCG NFYTQVPTRF RGGNQTKQDL 321ELHELVKLLR GKLRNTLKNC SEINTADGLF LEAASNFNII 361QEDLEDEQVD VRIFTTLCRM PLYETEFGWG KPEWVTIPEM 401HLEIVFLLDT KCGTGIEALV SMDEADMLQF ELDPTISAFA 441 S

Other nucleic acids encoding the feruloyl-CoA:monolignol transferasesthat are useful for making coniferyl ferulate (and other monolignolferulates) were isolated from the stem of Hibiscus cannabinus (Kenaf).The coding region of the Hibiscus cannabinus (Kenaf) has the followingnucleic acid sequence (SEQ ID NO:8).

1 ATGGCAACCC ACAGCACTAT CATGTTCTCA GTCGATAGAA 41ACGATGTCGT GTTTGTCAAA CCCTTCAAAC CTACACCCTC 81ACAGGTTCTA TCTCTCTCCA CCATCGACAA TGATCCCAAC 121CTTGAGATCA TGTGCCATAC TGTTTTTGTG TATCAAGCCA 161ATGCCGATTT CGATGTTAAG CCCAAGGATC CAGCTTCCAT 201AATCCAGGAA GCACTCTCCA AGCTCTTGGT TTATTACTAT 241CCCTTAGCGG GGAAGATGAA GAGGGAGACC GATGGAAAAC 281TTCGAATCGC TTGCACTGCC GACGATAGCG TGCCCTTCTT 321AGTAGCCACC GCCGATTGCA AGCTCTCGTC GTTGAACCAC 361TTGGATGGCA TAGATGTTCA TACCGGGAAA GAATTCGCCT 401TGGATTTTGC ATCCGAATCC GACGGTGGCT ATTATCACCC 441TCTGGTCATG CAGGTGACGA AGTTCATATG CGGAGGGTTC 481ACCATCGCTT TGAGTTTATC GCACTCGGTT TGTGATGGCT 521TCGGTGCAGC TCAGATCTTT CAAGCATTGA CCGAGCTCGC 561AAGTGGCAGG AACGAGCCCT CGGTTAAACC CGTGTGGGAG 601AGGCAACTAT TAGTGGCGAA ACCGGCCGAG GAAATCCCTC 641GGTCGATTGT CGATAAGGAC TTGTCGGCAG CTTCACCGTA 681TCTGCCGACA ACCGACATAG TCCATGCCTG CTTTTATGTA 721ACCGAGGAGA GTATAAAAAC ACTGAAAATG AATCTGATCA 761AAGAAAGCAA AGATGAGAGT ATAACCAGTC TCGAGGTCCT 801TTCAGCCTAT ATATGGAGAG CAAGGTTTAG AGCATTGAAA 841TTGAGTCCAG ATAAAACCAC AATGCTCGGC ATGGCCGTAG 881GCATACGACG CACCGTGAAA CCACGGTTGC CCGAAGGATA 921CTACGGGAAT GCTTTCACCT CGGCAAATAC GGCCATGACC 961GGGAAGGAAC TCGACCAAGG ACCGCTCTCG AAAGCTGTGA 1001AACAAATCAA GGAGAGCAAA AAGCTTGCTT CGGAGAATGA 1041CTATATCTGG AACTTGATGA GCATTAACGA GAAGCTGAGA 1081GAACTGAATT CGAAGTTCGA AGCGGCCGCC GGTTCAACCA 1121TGGTCATAAC AGATTGGAGG CGGTTGGGAC TATTGGAAGA 1161TGTGGATTTT GGATGGAAAG GTAGCGTAAA CATGATACCA 1201CTGCCGTGGA ACATGTTCGG GTACGTGGAT TTGGTTCTTT 1241TATTGCCTCC TTGTAAACTG GACCAATCGA TGAAAGGCGG 1281TGCTAGAGTG TTGGTTTCCT TTCCCACGGC TGCTATTGCC 1321AAATTCAAGG AAGAAATGGA TGCTCTCAAA CATGATAACA 1361AGGTTGCCGG CGATGCTCTA GTGATCTAG

The SEQ ID NO:8 nucleic acid encodes a Hibiscus cannabinus (Kenaf).feruloyl-CoA:monolignol transferase enzyme with the following amino acidsequence (SEQ ID NO:9).

1 MATHSTIMFS VDRNDVVFVK PFKPTPSQVL SLSTIDNDPN 41LEIMCHTVFV YQANADFDVK PKDPASIIQE ALSKLLVYYY 81PLAGKMKRET DGKLRIACTA DDSVPFLVAT ADCKLSSLNH 121LDGIDVHTGK EFALDFASES DGGYYHPLVM QVTKFICGGF 161TIALSLSHSV CDGFGAAQIF QALTELASGR NEPSVKPVWE 201RQLLVAKPAE EIPRSIVDKD LSAASPYLPT TDIVHACFYV 241TEESIKTLKM NLIKESKDES ITSLEVLSAY IWRARFRALK 281LSPDKTTMLG MAVGIRRTVK PRLPEGYYGN AFTSANTAMT 321GKELDQGPLS KAVKQIKESK KLASENDYIW NLMSINEKLR 361ELNSKFEAAA GSTMVITDWR RLGLLEDVDF GWKGSVNMIP 401LPWNMFGYVD LVLLLPPCKL DQSMKGGARV LVSFPTAAIA 441 KFKEEMDALK HDNKVAGDAL VI

The SEQ ID NO:8 nucleic acid also encodes a Hibiscus cannabinus (Kenaf)feruloyl-CoA:monolignol transferase enzyme with the SEQ ID NO:16 aminoacid sequence shown below.

70                                E ALSKLLVYYY 81PLAGKMKRET DGKLRIACTA DDSVPFLVAT ADCKLSSLNH 121LDGIDVHTGK EFALDFASES DGGYYHPLVM QVTKFICGGF 161TIALSLSHSV CDGFGAAQIF QALTELASGR NEPSVKPVWE 201RQLLVAKPAE EIPRSIVDKD LSAASPYLPT TDIVHACFYV 241TEESIKTLKM NLIKESKDES ITSLEVLSAY IWRARFRALK 281LSPDKTTMLG MAVGIRRTVK PRLPEGYYGN AFTSANTAMT 321GKELDQGPLS KAVKQIKESK KLASENDYIW NLMSINEKLR 361ELNSKFEAAA GSTMVITDWR RLGLLEDVDF GWKGSVNMIP 401LPWNMFGYVD LVLLLPPCKL DQSMKGGARV LVSFPTAAIA 441 KFK

Nucleic acids encoding this new class of BAHD acyltransferases allowidentification and isolation of related nucleic acids and their encodedenzymes that provide a means for production of altered lignins inplants.

For example, related nucleic acids can be isolated and identified bymutation of the SEQ ID NO:8 sequence and/or by hybridization to DNAand/or RNA isolated from other plant species using SEQ ID NO:8 nucleicacids as probes. The sequence of the feruloyl-CoA:monolignol transferaseenzyme (e.g., SEQ ID NO:9 or SEQ ID NO:16) can also be examined and useda basis for designing alternative feruloyl-CoA:monolignol transferasenucleic acids that encode related feruloyl-CoA:monolignol transferasepolypeptides.

In one embodiment, the BAHD acyltransferase nucleic acids of theinvention include any nucleic acid that can selectively hybridize to SEQID NO:8.

The term “selectively hybridize” includes hybridization, under stringenthybridization conditions, of a nucleic acid sequence to a specifiednucleic acid target sequence (e.g., SEQ ID NO:8) to a detectably greaterdegree (e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences. Such selective hybridizationsubstantially excludes non-target nucleic acids. Selectively hybridizingsequences typically have about at least 40% sequence identity, or atleast 50% sequence identity, or at least 60% sequence identity, or atleast 70% sequence identity, or 60-99% sequence identity, or 70-99%sequence identity, or 80-99% sequence identity, or 90-95% sequenceidentity, or 90-99% sequence identity, or 95-97% sequence identity, or97-99% sequence identity, or 100% sequence identity (or complementarity)with each other. In some embodiments, a selectively hybridizing sequencehas at least about 70% or at least about 80% sequence identity orcomplementarity with SEQ ID NO:8.

Thus, the nucleic acids of the invention include those with about 500 ofthe same nucleotides as SEQ ID NO:8, or about 600 of the samenucleotides as SEQ ID NO:8, or about 700 of the same nucleotides as SEQID NO:8, or about 800 of the same nucleotides as SEQ ID NO:8, or about900 of the same nucleotides as SEQ ID NO:8, or about 1000 of the samenucleotides as SEQ ID NO:8, or about 1100 of the same nucleotides as SEQID NO:8, or about 1200 of the same nucleotides as SEQ ID NO:8, or about1300 of the same nucleotides as SEQ ID NO:8, or about 500-1325 of thesame nucleotides as SEQ ID NO:8. The identical nucleotides or aminoacids can be distributed throughout the nucleic acid or the protein, andneed not be contiguous.

Note that if a value of a variable that is necessarily an integer, e.g.,the number of nucleotides or amino acids in a nucleic acid or protein,is described as a range, e.g., 90-99% sequence identity what is meant isthat the value can be any integer between 90 and 99 inclusive, i.e., 90,91, 92, 93, 94, 95, 96, 97, 98 or 99, or any range between 90 and 99inclusive, e.g., 91-99%, 91-98%, 92-99%, etc.

The terms “stringent conditions” or “stringent hybridization conditions”include conditions under which a probe will hybridize to its targetsequence to a detectably greater degree than other sequences (e.g., atleast 2-fold over background). Stringent conditions are somewhatsequence-dependent and can vary in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences can be identified with up to 100%complementarity to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of sequence similarity are detected(heterologous probing). The probe can be approximately 20-500nucleotides in length, but can vary greatly in length from about 18nucleotides to equal to the entire length of the target sequence. Insome embodiments, the probe is about 10-50 nucleotides in length, orabout 18-25 nucleotides in length, or about 18-50 nucleotides in length,or about 18-100 nucleotides in length.

Typically, stringent conditions will be those where the saltconcentration is less than about 1.5 M Na ion (or other salts),typically about 0.01 to 1.0 M Na ion concentration (or other salts), atpH 7.0 to 8.3 and the temperature is at least about 30° C. for shorterprobes (e.g., 10 to 50 nucleotides) and at least about 60° C. for longerprobes (e.g., greater than 50 nucleotides). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide or Denhardt's solution. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1MNaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and a wash in 1×SSC to2×SSC (where 20×SSC is 3.0 M NaCl, 0.3 M trisodium citrate) at 50 to 55°C. Exemplary moderate stringency conditions include hybridization in 40to 45% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in 0.5×SSC to1×SSC at 55 to 60° C. Exemplary high stringency conditions includehybridization in 50% formamide, 1M NaCl, 1% SDS at 37° C., and a wash in0.1×SSC at 60 to 65° C. Specificity is typically a function ofpost-hybridization washes, where the factors controlling hybridizationinclude the ionic strength and temperature of the final wash solution.

For DNA-DNA hybrids, the T_(m) can be approximated from the equation ofMeinkoth and Wahl (Anal. Biochem. 138:267-84 (1984)):

T _(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% formamide)−500/L

where M is the molarity of monovalent cations; % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % formamide is thepercentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. The T_(m), is reducedby about 1° C. for each 1% of mismatching. Thus, the T_(m),hybridization and/or wash conditions can be adjusted to hybridize tosequences of the desired sequence identity. For example, if sequenceswith greater than or equal to 90% sequence identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can include hybridizationand/or a wash at 1, 2, 3 or 4° C. lower than the thermal melting point(T_(m)). Moderately stringent conditions can include hybridizationand/or a wash at 6, 7, 8, 9 or 10° C. lower than the thermal meltingpoint (T_(m)). Low stringency conditions can include hybridizationand/or a wash at 11, 12, 13, 14, 15 or 20° C. lower than the thermalmelting point (T_(m)). Using the equation, hybridization and washcompositions, and a desired T_(m), those of ordinary skill can identifyand isolate nucleic acids with sequences related to SEQ ID NO:8.

Those of skill in the art also understand how to vary the hybridizationand/or wash solutions to isolate desirable nucleic acids. For example,if the desired degree of mismatching results in a T_(m) of less than 45°C. (aqueous solution) or 32° C. (formamide solution) it is preferred toincrease the SSC concentration so that a higher temperature can be used.

An extensive guide to the hybridization of nucleic acids is found inTijssen, LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY—HYBRIDIZATION WITH NUCLEIC ACID PROBES, part 1, chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” Elsevier, N.Y. (1993); and in CURRENT PROTOCOLS INMOLECULAR BIOLOGY, chapter 2, Ausubel, et al., eds, Greene Publishingand Wiley-Interscience, New York (1995).

Unless otherwise stated, in the present application high stringency isdefined as hybridization in 4×SSC, 5×Denhardt's (5 g Ficoll, 5 gpolyvinypyrrolidone, 5 g bovine serum albumin in 500 ml of water), 0.1mg/ml boiled salmon sperm DNA, and 25 mM Na phosphate at 65° C., and awash in 0.1×SSC, 0.1% SDS at 65° C.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polypeptides: (a) “referencesequence,” (b) “comparison window,” (c) “sequence identity,” (d)“percentage of sequence identity” and (e) “substantial identity.”

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. The reference sequence can be a nucleicacid sequence (e.g., SEQ ID NO:8) or an amino acid sequence (e.g., SEQID NO:9 or 16). A reference sequence may be a subset or the entirety ofa specified sequence. For example, a reference sequence may be a segmentof a full-length cDNA or of a genomic DNA sequence, or the complete cDNAor complete genomic DNA sequence, or a domain of a polypeptide sequence.

As used herein, “comparison window” refers to a contiguous and specifiedsegment of a nucleic acid or an amino acid sequence, wherein the nucleicacid/amino acid sequence can be compared to a reference sequence andwherein the portion of the nucleic acid/amino acid sequence in thecomparison window may comprise additions or deletions (i.e., gaps)compared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. The comparisonwindow can vary for nucleic acid and polypeptide sequences. Generally,for nucleic acids, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100 or morenucleotides. For amino acid sequences, the comparison window is at leastabout 10 amino acids, and can optionally be 15, 20, 30, 40, 50, 100 ormore amino acids. Those of skill in the art understand that to avoid ahigh similarity to a reference sequence due to inclusion of gaps in thenucleic acid or amino acid sequence, a gap penalty is typicallyintroduced and is subtracted from the number of matches.

Methods of alignment of nucleotide and amino acid sequences forcomparison are well known in the art. The local homology algorithm(BESTFIT) of Smith and Waterman, (1981) Adv. Appl. Math 2:482, maypermit optimal alignment of compared sequences; by the homologyalignment algorithm (GAP) of Needleman and Wunsch, (1970) J. Mol. Biol.48:443-53; by the search for similarity method (Tfasta and Fasta) ofPearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444; bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif., GAP, BESTFIT, BLAST, FASTA and TFASTA in the WisconsinGenetics Software Package, Version 8 (available from Genetics ComputerGroup (GCG™ programs (Accelrys, Inc., San Diego, Calif.)). The CLUSTALprogram is well described by Higgins and Sharp (1988) Gene 73:237-44;Higgins and Sharp, (1989) CABIOS 5:151-3; Corpet, et al., (1988) NucleicAcids Res. 16:10881-90; Huang, et al., (1992) Computer Applications inthe Biosciences 8:155-65 and Pearson, et al., (1994) Meth. Mol. Biol.24:307-31. An example of a good program to use for optimal globalalignment of multiple sequences is PileUp (Feng and Doolittle, (1987) J.Mol. Evol., 25:351-60, which is similar to the method described byHiggins and Sharp, (1989) CABIOS 5:151-53 (and is hereby incorporated byreference). The BLAST family of programs that can be used for databasesimilarity searches includes: BLASTN for nucleotide query sequencesagainst nucleotide database sequences; BLASTX for nucleotide querysequences against protein database sequences; BLASTP for protein querysequences against protein database sequences; TBLASTN for protein querysequences against nucleotide database sequences; and TBLASTX fornucleotide query sequences against nucleotide database sequences. See,Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al.,eds., Greene Publishing and Wiley-Interscience, New York (1995).

GAP uses the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443-53, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP makes a profit of gapcreation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the Wisconsin GeneticsSoftware Package are 8 and 2, respectively. The gap creation and gapextension penalties can be expressed as an integer selected from thegroup of integers consisting of from 0 to 100. Thus, for example, thegap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 30, 40, 50 or more.

GAP presents one member of the family of best alignments. There may bemany members of this family. GAP displays four figures of merit foralignments: Quality, Ratio, Identity and Similarity. The Quality is themetric maximized in order to align the sequences. Ratio is the qualitydivided by the number of bases in the shorter segment. Percent Identityis the percent of the symbols that actually match. Percent Similarity isthe percent of the symbols that are similar. Symbols that are acrossfrom gaps are ignored. A similarity is scored when the scoring matrixvalue for a pair of symbols is greater than or equal to 0.50, thesimilarity threshold. The scoring matrix used in Version 10 of theWisconsin Genetics Software Package is BLOSUM62 (see, Henikoff andHenikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915).

For example, sequence identity/similarity values provided herein canrefer to the value obtained using the BLAST 2.0 suite of programs usingdefault parameters (Altschul, et al., (1997) Nucleic Acids Res.25:3389-402).

As those of ordinary skill in the art will understand, BLAST searchesassume that proteins can be modeled as random sequences. However, manyreal proteins comprise regions of nonrandom sequences, which may behomopolymeric tracts, short-period repeats, or regions enriched in oneor more amino acids. Such low-complexity regions may be aligned betweenunrelated proteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs can be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen, (1993) Comput. Chem. 17:149-63) and XNU (C.sub.1-ayerieand States, (1993) Comput. Chem. 17:191-201) low-complexity filters canbe employed alone or in combination.

The terms “substantial identity” indicates that a polypeptide or nucleicacid comprises a sequence with between 55-100% sequence identity to areference sequence, with at least 55% sequence identity, or at least60%, or at least 70%, or at least 80%, or at least 90% or at least 95%sequence identity, or any percentage value within the range of 55-100%sequence identity relative to the reference sequence over a specifiedcomparison window. Optimal alignment may be ascertained or conductedusing the homology alignment algorithm of Needleman and Wunsch, supra.

An indication that two polypeptide sequences are substantially identicalis that both polypeptides have feruloyl-CoA:monolignol transferaseactivity, meaning that both polypeptides can synthesize monolignolferulates from a monolignol and feruloyl-CoA. The polypeptide that issubstantially identical to a feruloyl-CoA:monolignol transferase with aSEQ ID NO:9 or 16 sequence may not have exactly the same level ofactivity as the feruloyl-CoA:monolignol transferase with a SEQ ID NO:9or 16. Instead, the substantially identical polypeptide may exhibitgreater or lesser levels of feruloyl-CoA:monolignol transferase activitythan the feruloyl-CoA:monolignol transferase with SEQ ID NO:9 or 16, asmeasured by assays available in the art or described herein (see, e.g.,Example 1). For example, the substantially identical polypeptide canhave at least about 40%, or at least about 50%, or at least about 60%,or at least about 70%, or at least about 80%, or at least about 90%, orat least about 95%, or at least about 97%, or at least about 98%, or atleast about 100%, or at least about 105%, or at least about 110%, or atleast about 120%, or at least about 130%, or at least about 140%, or atleast about 150%, or at least about 200% of the activity of theferuloyl-CoA:monolignol transferase with the SEQ ID NO:9 or 16 sequencewhen measured by similar assay procedures.

Alternatively, substantial identity is present when second polypeptideis immunologically reactive with antibodies raised against the firstpolypeptide (e.g., a polypeptide with SEQ ID NO:9 or 16). Thus, apolypeptide is substantially identical to a first polypeptide, forexample, where the two polypeptides differ only by a conservativesubstitution. In addition, a polypeptide can be substantially identicalto a first polypeptide when they differ by a non-conservative change ifthe epitope that the antibody recognizes is substantially identical.Polypeptides that are “substantially similar” share sequences as notedabove except that some residue positions, which are not identical, maydiffer by conservative amino acid changes.

The feruloyl-CoA:monolignol transferase polypeptides of the presentinvention may include the first 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99 N-terminalamino acid residues of a the SEQ ID NO:9 or 16 sequence. Alternatively,the feruloyl-CoA:monolignol transferase polypeptides of the presentinvention may include the first 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99 C-terminalamino acid residues of a the SEQ ID NO:9 or 16 sequence.

Lignin

Lignin broadly refers to a biopolymer that is typically part ofsecondary cell walls in plants. Lignin is a complex moderatelycross-linked aromatic polymer (see, e.g., FIG. 1). Lignin may also becovalently linked to hemicelluloses. Hemicellulose broadly refers to aclass of branched sugar polymers composed of pentoses and hexoses.Hemicelluloses typically have an amorphous structure with up to hundredsor thousands of pentose units and they are generally at least partiallysoluble in dilute alkali. Cellulose broadly refers to an organiccompound with the formula (C₆H₁₀O₅)_(z) where z is an integer. Celluloseis a linear polysaccharide that can include linear chains ofbeta-1-4-linked glucose residues of several hundred to over ten thousandunits.

Lignocellulosic biomass represents an abundant, inexpensive, and locallyavailable feedstock for conversion to carbonaceous fuel (e.g., ethanol,biodiesel, biofuel and the like). However, the complex structure oflignin, which includes ether and carbon-carbon bonds that bind togetherthe various subunits of lignin, and the crosslinking of lignin to otherplant cell wall polymers, make it the most recalcitrant of plantpolymers. Thus, significant quantities of lignin in a biomass caninhibit the efficient usage of plants as a source of fuels and othercommercial products. Gaining access to the carbohydrate andpolysaccharide polymers of plant cells for use as carbon and energysources therefore requires significant energy input and often harshchemical treatments, especially when significant amounts of lignin arepresent. For example, papermaking procedures in which lignin is removedfrom plant fibers by delignification reactions are typically expensive,can be polluting and generally require use of high temperatures andharsh chemicals largely because the structure of lignin is impervious tomild conditions. Plants with altered lignin structures that could bemore readily cleaved under milder conditions would reduce the costs ofpapermaking and make the production of biofuels more competitive withcurrently existing procedures for producing oil and gas fuels.

Plants make lignin from a variety of subunits or monomers that aregenerally termed monolignols. Such primary monolignols includep-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol.

Monolignols destined for lignin polymerization in normal plants can bepreacylated with acetate, p-hydroxybenzoate, or p-coumarate (Ralph etal., Phytochem. Rev. 3:29-60 (2004)). p-Coumarates can acylate theγ-position of phenylpropanoid side chains mainly found in the syringylunits of lignin. Studies indicate that monolignols, primarily sinapylalcohol, are enzymatically pre-acylated with p-coumarate prior to theirincorporation into lignin, indicating that the monolignolp-coumarateconjugates, coniferyl p-coumarate and sinapyl p-coumarate, can also be‘monomer’ precursors of lignin.

While monolignolp-coumarate-derived units may comprise up to 40% of thelignin in some grass tissues, the p-coumarate moiety from suchconjugates does not enter into the radical coupling (polymerization)reactions occurring during lignifications. Instead, the p-coumaratemoieties substantially remain as terminal units with an unsaturated sidechain and a free phenolic group (Ralph et al., J. Am. Chem. Soc. 116:9448-9456 (1994); Hatfield et al., J. Sci. Food Agric. 79: 891-899(1999)). Thus, the presence of sinapyl p-coumarate conjugates produces alignin ‘core’ with terminal p-coumarate groups and no new bonds in thebackbone of the lignin polymer, resulting in a lignin that is notsignificantly more easily cleaved.

In contrast to p-coumarate, ferulate esters do undergo radical couplingreactions under lignification conditions. Model ferulates, such as theferulate shown below (where R is CH₃—, CH₃—CH₂—, a sugar, apolysaccharide, pectin, cell-wall (arabino)xylan or other plantcomponent), readily undergo radical coupling reactions with each otherand with lignin monomers and oligomers to form cross-linked networks.

If present during lignification, ferulates can become inextricably boundinto the lignin by ether and C—C bonds. Although such ferulate moietiesare no more extractable or cleavable from the lignin structure thanother lignin units under most conditions, the ester itself can bereadily cleaved using conditions generally employed for ester cleavage.Upon cleavage of such ester bonds, other plant cell wall components canbe released. For example, an arabinoxylan (hemicellulose) chain can bereleased from a ferulate-mediated lignin attachment by cleaving theester.

Ferulate-monolignol ester conjugates (unlike their p-coumarate analogs),such as coniferyl ferulate or sinapyl ferulate have not been identifiedin natural plant lignins, but some types of plants make them assecondary metabolites during, among other things, lignin biosynthesis.[Paula et al, Tetrahedron 51: 12453-12462 (1994); Seca et al.,Phytochemistry 56: 759-767 (2001); Hsiao & Chiang, Phytochemistry 39:899-902 (1995); Li et al., Planta Med. 72: 278-280 (2005)]. Thestructures of coniferyl ferulate and sinapyl ferulate are shown below.

For example, the feruloyl-CoA:monolignol transferases provided hereinbiosynthesize coniferyl ferulate from coniferyl alcohol and feruloyl-CoAas shown below.

The incorporation of monolignol ferulates into the lignin of plantsallows the cell wall materials and lignin to be readily cleaved orprocessed into useful products. See also, U.S. Patent Application No.61/213,706, the contents of which are specifically incorporated hereinby reference in their entirety.

The monolignol ferulates made by the methods and feruloyl-CoA:monolignoltransferases provided herein can be incorporated by radical couplinginto plant lignins. Both the monolignol and the ferulate moieties canundergo such coupling, resulting in a lignin that can be complex.However, such ‘double-ended-incorporation’ still yields readilycleavable ester linkages that have been engineered into the backbone ofthe lignin polymer network. Esters are readily cleaved under much lessstringent conditions by the same chemical processes used to cleavelignin, but the lignin resulting from the methods described herein issignificantly easier to cleave, and provides more facile and less costlyaccess to the plant cell wall polysaccharides. See also, “Method formodifying lignin structure using monolignol ferulate conjugates”, U.S.Patent Application No. 61/213,706.

Lignins can be degraded by chemical or enzymatic means to yield avariety of smaller monomers and oligomers. While enzymatic processes aregenerally preferred because they do not require high temperatures andharsh chemicals, such enzymatic processes have previously not been aseffective at solubilizing lignin moieties away from valuable plant cellconstituents (e.g., polysaccharides and carbohydrates).

According to the invention, plants with the feruloyl-CoA:monolignoltransferase nucleic acids and/or enzymes described herein supplymonolignol ferulates for facile lignification in plants, therebyyielding plants with lignins that are more readily cleaved or processedto release cellulose, hemicelluloses and lignin breakdown products.

Conditions for releasing the cellulose, hemicelluloses and ligninbreakdown products from plants containing the feruloyl-CoA:monolignoltransferase nucleic acids and/or enzymes described herein includeconditions typically employed for cleaving ester bonds. Thus, the esterbonds within monolignol ferulate-rich lignins can be cleaved by milderalkaline and/or acidic conditions than the conditions typically used tobreak down the lignin of plants that are not rich in monolignolferulates. For example, mildly alkaline conditions involving use ofammonia may be used to cleave the ester bonds within monolignolferulate-rich lignins, whereas such conditions would not cleavesubstantially any of the ether and carbon-carbon bonds in normallignins. See also, PCT/US2011/044981 filed Jul. 22, 2011,PCT/US2011/045044 filed Jul. 22, 2011, and U.S. patent application Ser.No. 12/830,905, filed Jul. 6, 2010, the contents of both of which arespecifically incorporated herein by reference in their entireties.

Plants Modified to Contain a Feruloyl-CoA:Monolignol Transferase

In order to engineer plants with lignins that contain significant levelsof monolignol ferulates, one of skill in the art can introduceferuloyl-CoA:monolignol transferases or nucleic acids encoding suchferuloyl-CoA:monolignol transferases into the plants.

For example, one of skill in the art can inject feruloyl-CoA:monolignoltransferase enzymes into young plants.

Alternatively, one of skill in the art can generate genetically-modifiedplants that contain nucleic acids encoding feruloyl-CoA:monolignoltransferases within their somatic and/or germ cells. Such geneticmodification can be accomplished by procedures available in the art. Forexample, one of skill in the art can prepare an expression cassette orexpression vector that can express one or more encodedferuloyl-CoA:monolignol transferase enzymes. Plant cells can betransformed by the expression cassette or expression vector, and wholeplants (and their seeds) can be generated from the plant cells that weresuccessfully transformed with the feruloyl-CoA:monolignol transferasenucleic acids. Some procedures for making such genetically modifiedplants and their seeds are described below.

Promoters:

The feruloyl-CoA:monolignol transferase nucleic acids of the inventioncan be operably linked to a promoter, which provides for expression ofmRNA from the feruloyl-CoA:monolignol transferase nucleic acids. Thepromoter is typically a promoter functional in plants and/or seeds, andcan be a promoter functional during plant growth and development. Aferuloyl-CoA:monolignol transferase nucleic acid is operably linked tothe promoter when it is located downstream from the promoter, to therebyform an expression cassette.

Most endogenous genes have regions of DNA that are known as promoters,which regulate gene expression. Promoter regions are typically found inthe flanking DNA upstream from the coding sequence in both prokaryoticand eukaryotic cells. A promoter sequence provides for regulation oftranscription of the downstream gene sequence and typically includesfrom about 50 to about 2,000 nucleotide base pairs. Promoter sequencesalso contain regulatory sequences such as enhancer sequences that caninfluence the level of gene expression. Some isolated promoter sequencescan provide for gene expression of heterologous DNAs, that is a DNAdifferent from the native or homologous DNA.

Promoter sequences are also known to be strong or weak, or inducible. Astrong promoter provides for a high level of gene expression, whereas aweak promoter provides for a very low level of gene expression. Aninducible promoter is a promoter that provides for the turning on andoff of gene expression in response to an exogenously added agent, or toan environmental or developmental stimulus. For example, a bacterialpromoter such as the P_(tac) promoter can be induced to vary levels ofgene expression depending on the level of isothiopropylgalactoside addedto the transformed cells. Promoters can also provide for tissue specificor developmental regulation. An isolated promoter sequence that is astrong promoter for heterologous DNAs is advantageous because itprovides for a sufficient level of gene expression for easy detectionand selection of transformed cells and provides for a high level of geneexpression when desired.

Expression cassettes generally include, but are not limited to, a plantpromoter such as the CaMV 35S promoter (Odell et al., Nature.313:810-812 (1985)), or others such as CaMV 19S (Lawton et al., PlantMolecular Biology. 9:315-324 (1987)), nos (Ebert et al., Proc. Natl.Acad. Sci. USA. 84:5745-5749 (1987)), Adhl (Walker et al., Proc. Natl.Acad. Sci. USA. 84:6624-6628 (1987)), sucrose synthase (Yang et al.,Proc. Natl. Acad. Sci. USA. 87:4144-4148 (1990)), α-tubulin, ubiquitin,actin (Wang et al., Mol. Cell. Biol. 12:3399 (1992)), cab (Sullivan etal., Mol. Gen. Genet. 215:431 (1989)), PEPCase (Hudspeth et al., PlantMolecular Biology. 12:579-589 (1989)) or those associated with the Rgene complex (Chandler et al., The Plant Cell. 1:1175-1183 (1989)).Further suitable promoters include the poplar xylem-specific secondarycell wall specific cellulose synthase 8 promoter, cauliflower mosaicvirus promoter, the Z10 promoter from a gene encoding a 10 kD zeinprotein, a Z27 promoter from a gene encoding a 27 kD zein protein,inducible promoters, such as the light inducible promoter derived fromthe pea rbcS gene (Coruzzi et al., EMBO J. 3:1671 (1971)) and the actinpromoter from rice (McElroy et al., The Plant Cell. 2:163-171 (1990)).Seed specific promoters, such as the phaseolin promoter from beans, mayalso be used (Sengupta-Gopalan, Proc. Natl. Acad. Sci. USA. 83:3320-3324(1985). Other promoters useful in the practice of the invention areknown to those of skill in the art.

Alternatively, novel tissue specific promoter sequences may be employedin the practice of the present invention. cDNA clones from a particulartissue can be isolated and those clones which are expressed specificallyin that tissue are identified, for example, using Northern blotting.Preferably, the gene isolated is not present in a high copy number, butis relatively abundant in specific tissues. The promoter and controlelements of corresponding genomic clones can then be localized usingtechniques well known to those of skill in the art.

A feruloyl-CoA:monolignol transferase nucleic acid can be combined withthe promoter by standard methods to yield an expression cassette, forexample, as described in Sambrook et al. (MOLECULAR CLONING: ALABORATORY MANUAL. Second Edition (Cold Spring Harbor, N.Y.: Cold SpringHarbor Press (1989); MOLECULAR CLONING: A LABORATORY MANUAL. ThirdEdition (Cold Spring Harbor, N.Y.: Cold Spring Harbor Press (2000)).Briefly, a plasmid containing a promoter such as the 35S CaMV promotercan be constructed as described in Jefferson (Plant Molecular BiologyReporter 5:387-405 (1987)) or obtained from Clontech Lab in Palo Alto,Calif. (e.g., pBI121 or pBI221). Typically, these plasmids areconstructed to have multiple cloning sites having specificity fordifferent restriction enzymes downstream from the promoter. Theferuloyl-CoA:monolignol transferase nucleic acids can be subcloneddownstream from the promoter using restriction enzymes and positioned toensure that the DNA is inserted in proper orientation with respect tothe promoter so that the DNA can be expressed as sense RNA. Once theferuloyl-CoA:monolignol transferase nucleic acid is operably linked to apromoter, the expression cassette so formed can be subcloned into aplasmid or other vector (e.g., an expression vector).

In some embodiments, a cDNA clone encoding a feruloyl-CoA:monolignoltransferase protein is isolated from Hibiscus cannabinus tissue, forexample, a root tissue. In other embodiments, cDNA clones from otherspecies (that encode a feruloyl-CoA:monolignol transferase protein) areisolated from selected plant tissues, or a nucleic acid encoding amutant or modified feruloyl-CoA:monolignol transferase protein isprepared by available methods or as described herein. For example, thenucleic acid encoding a mutant or modified feruloyl-CoA:monolignoltransferase protein can be any nucleic acid with a coding region thathybridizes to SEQ ID NO:8 and that has feruloyl-CoA:monolignoltransferase activity. Using restriction endonucleases, the entire codingsequence for the feruloyl-CoA:monolignol transferase is subcloneddownstream of the promoter in a 5′ to 3′ sense orientation.

Targeting Sequences:

Additionally, expression cassettes can be constructed and employed totarget the feruloyl-CoA:monolignol transferase nucleic acids to anintracellular compartment within plant cells or to direct an encodedprotein to the extracellular environment. This can generally be achievedby joining a DNA sequence encoding a transit or signal peptide sequenceto the coding sequence of the feruloyl-CoA:monolignol transferasenucleic acid. The resultant transit, or signal, peptide will transportthe protein to a particular intracellular, or extracellular destination,respectively, and can then be posttranslational removed. Transitpeptides act by facilitating the transport of proteins throughintracellular membranes, e.g., vacuole, vesicle, plastid andmitochondrial membranes, whereas signal peptides direct proteins throughthe extracellular membrane. By facilitating transport of the proteininto compartments inside or outside the cell, these sequences canincrease the accumulation of a particular gene product in a particularlocation. For example, see U.S. Pat. No. 5,258,300.

3′ Sequences:

When the expression cassette is to be introduced into a plant cell, theexpression cassette can also optionally include 3′ nontranslated plantregulatory DNA sequences that act as a signal to terminate transcriptionand allow for the polyadenylation of the resultant mRNA. The 3′nontranslated regulatory DNA sequence preferably includes from about 300to 1,000 nucleotide base pairs and contains plant transcriptional andtranslational termination sequences. For example, 3′ elements that canbe used include those derived from the nopaline synthase gene ofAgrobacterium tumefaciens (Bevan et al., Nucleic Acid Research.11:369-385 (1983)), or the terminator sequences for the T7 transcriptfrom the octopine synthase gene of Agrobacterium tumefaciens, and/or the3′ end of the protease inhibitor I or II genes from potato or tomato.Other 3′ elements known to those of skill in the art can also beemployed. These 3′ nontranslated regulatory sequences can be obtained asdescribed in An (Methods in Enzymology. 153:292 (1987)). Many such 3′nontranslated regulatory sequences are already present in plasmidsavailable from commercial sources such as Clontech, Palo Alto, Calif.The 3′ nontranslated regulatory sequences can be operably linked to the3′ terminus of the feruloyl-CoA:monolignol transferase nucleic acids bystandard methods.

Selectable and Screenable Marker Sequences:

In order to improve identification of transformants, a selectable orscreenable marker gene can be employed with the expressibleferuloyl-CoA:monolignol transferase nucleic acids. “Marker genes” aregenes that impart a distinct phenotype to cells expressing the markergene and thus allow such transformed cells to be distinguished fromcells that do not have the marker. Such genes may encode either aselectable or screenable marker, depending on whether the marker confersa trait which one can ‘select’ for by chemical means, i.e., through theuse of a selective agent (e.g., a herbicide, antibiotic, or the like),or whether it is simply a trait that one can identify throughobservation or testing, i.e., by ‘screening’ (e.g., the R-locus trait).Of course, many examples of suitable marker genes are known to the artand can be employed in the practice of the invention.

Included within the terms selectable or screenable marker genes are alsogenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which encode a secretable antigen that can be identifiedby antibody interaction, or secretable enzymes that can be detected bytheir catalytic activity. Secretable proteins fall into a number ofclasses, including small, diffusible proteins detectable, e.g., byELISA; and proteins that are inserted or trapped in the cell wall (e.g.,proteins that include a leader sequence such as that found in theexpression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene thatencodes a polypeptide that becomes sequestered in the cell wall, wherethe polypeptide includes a unique epitope may be advantageous. Such asecreted antigen marker can employ an epitope sequence that wouldprovide low background in plant tissue, a promoter-leader sequence thatimparts efficient expression and targeting across the plasma membrane,and can produce protein that is bound in the cell wall and yet isaccessible to antibodies. A normally secreted wall protein modified toinclude a unique epitope would satisfy such requirements.

Example of proteins suitable for modification in this manner includeextensin or hydroxyproline rich glycoprotein (HPRG). For example, themaize HPRG (Stiefel et al., The Plant Cell. 2:785-793 (1990)) is wellcharacterized in terms of molecular biology, expression, and proteinstructure and therefore can readily be employed. However, any one of avariety of extensins and/or glycine-rich wall proteins (Keller et al.,EMBO J. 8:1309-1314 (1989)) could be modified by the addition of anantigenic site to create a screenable marker.

Numerous other possible selectable and/or screenable marker genes willbe apparent to those of skill in the art in addition to those forthherein below. Therefore, it will be understood that the discussionherein is exemplary rather than exhaustive. In light of the techniquesdisclosed herein and the general recombinant techniques that are knownin the art, the present invention readily allows the introduction of anygene, including marker genes, into a recipient cell to generate atransformed plant cell, e.g., a monocot cell or dicot cell.

Possible selectable markers for use in connection with the presentinvention include, but are not limited to, a neo gene (Potrykus et al.,Mol. Gen. Genet. 199:183-188 (1985)) which codes for kanamycinresistance and can be selected for using kanamycin, G418, and the like;a bar gene which codes for bialaphos resistance; a gene which encodes analtered EPSP synthase protein (Hinchee et al., Bio/Technology. 6:915-922(1988)) thus conferring glyphosate resistance; a nitrilase gene such asbxn from Klebsiella ozaenae which confers resistance to bromoxynil(Stalker et al., Science. 242:419-423 (1988)); a mutant acetolactatesynthase gene (ALS) which confers resistance to imidazolinone,sulfonylurea or other ALS-inhibiting chemicals (European PatentApplication 154,204 (1985)); a methotrexate-resistant DHFR gene (Thilletet al., J. Biol. Chem. 263:12500-12508 (1988)); a dalapon dehalogenasegene that confers resistance to the herbicide dalapon; or a mutatedanthranilate synthase gene that confers resistance to 5-methyltryptophan. Where a mutant EPSP synthase gene is employed, additionalbenefit may be realized through the incorporation of a suitablechloroplast transit peptide, CTP (European Patent Application 0 218 571(1987)).

An illustrative embodiment of a selectable marker gene capable of beingused in systems to select transformants is the gene that encode theenzyme phosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicus or the pat gene from Streptomycesviridochromogenes (U.S. Pat. No. 5,550,318). The enzyme phosphinothricinacetyl transferase (PAT) inactivates the active ingredient in theherbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutaminesynthetase, (Murakami et al., Mol. Gen. Genet. 205:42-50 (1986); Twellet al., Plant Physiol. 91:1270-1274 (1989)) causing rapid accumulationof ammonia and cell death. The success in using this selective system inconjunction with monocots was surprising because of the majordifficulties that have been reported in transformation of cereals(Potrykus, Trends Biotech. 7:269-273 (1989)).

Screenable markers that may be employed include, but are not limited to,a β-glucuronidase or uidA gene (GUS) that encodes an enzyme for whichvarious chromogenic substrates are known; an R-locus gene, which encodesa product that regulates the production of anthocyanin pigments (redcolor) in plant tissues (Dellaporta et al., In: Chromosome Structure andFunction: Impact of New Concepts, 18^(th) Stadler Genetics Symposium, J.P. Gustafson and R. Appels, eds. (New York: Plenum Press) pp. 263-282(1988)); a β-lactamase gene (Sutcliffe, Proc. Natl. Acad. Sci. USA.75:3737-3741 (1978)), which encodes an enzyme for which variouschromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci.USA. 80:1101 (1983)) which encodes a catechol dioxygenase that canconvert chromogenic catechols; an α-amylase gene (Ikuta et al.,Bio/technology 8:241-242 (1990)); a tyrosinase gene (Katz et al., J.Gen. Microbiol. 129:2703-2714 (1983)) which encodes an enzyme capable ofoxidizing tyrosine to DOPA and dopaquinone which in turn condenses toform the easily detectable compound melanin; a β-galactosidase gene,which encodes an enzyme for which there are chromogenic substrates; aluciferase (lux) gene (Ow et al., Science. 234:856-859.1986), whichallows for bioluminescence detection; or an aequorin gene (Prasher etal., Biochem. Biophys. Res. Comm. 126:1259-1268 (1985)), which may beemployed in calcium-sensitive bioluminescence detection, or a green oryellow fluorescent protein gene (Niedz et al., Plant Cell Reports.14:403 (1995)).

For example, genes from the maize R gene complex can be used asscreenable markers. The R gene complex in maize encodes a protein thatacts to regulate the production of anthocyanin pigments in most seed andplant tissue.

Maize strains can have one, or as many as four, R alleles that combineto regulate pigmentation in a developmental and tissue specific manner.A gene from the R gene complex does not harm the transformed cells.Thus, an R gene introduced into such cells will cause the expression ofa red pigment and, if stably incorporated, can be visually scored as ared sector. If a maize line carries dominant alleles for genes encodingthe enzymatic intermediates in the anthocyanin biosynthetic pathway (C2,A1, A2, Bz1 and Bz2), but carries a recessive allele at the R locus,transformation of any cell from that line with R will result in redpigment formation. Exemplary lines include Wisconsin 22 that containsthe rg-Stadler allele and TR112, a K55 derivative that is r-g, b, Pl.Alternatively any genotype of maize can be utilized if the C1 and Ralleles are introduced together.

The R gene regulatory regions may be employed in chimeric constructs inorder to provide mechanisms for controlling the expression of chimericgenes. More diversity of phenotypic expression is known at the R locusthan at any other locus (Coe et al., in Corn and Corn Improvement, eds.Sprague, G. F. & Dudley, J. W. (Am. Soc. Agron., Madison, Wis.), pp.81-258 (1988)). It is contemplated that regulatory regions obtained fromregions 5′ to the structural R gene can be useful in directing theexpression of genes, e.g., insect resistance, drought resistance,herbicide tolerance or other protein coding regions. For the purposes ofthe present invention, it is believed that any of the various R genefamily members may be successfully employed (e.g., P, S, Lc, etc.).However, one that can be used is Sn (particularly Sn:bol3). Sn is adominant member of the R gene complex and is functionally similar to theR and B loci in that Sn controls the tissue specific deposition ofanthocyanin pigments in certain seedling and plant cells, therefore, itsphenotype is similar to R.

A further screenable marker contemplated for use in the presentinvention is firefly luciferase, encoded by the lux gene. The presenceof the lux gene in transformed cells may be detected using, for example,X-ray film, scintillation counting, fluorescent spectrophotometry,low-light video cameras, photon counting cameras or multiwellluminometry. It is also envisioned that this system may be developed forpopulation screening for bioluminescence, such as on tissue cultureplates, or even for whole plant screening.

Other Optional Sequences:

An expression cassette of the invention can also further compriseplasmid DNA. Plasmid vectors include additional DNA sequences thatprovide for easy selection, amplification, and transformation of theexpression cassette in prokaryotic and eukaryotic cells, e.g.,pUC-derived vectors such as pUC8, pUC9, pUC18, pUC19, pUC23, pUC119, andpUC120, pSK-derived vectors, pGEM-derived vectors, pSP-derived vectors,or pBS-derived vectors. The additional DNA sequences include origins ofreplication to provide for autonomous replication of the vector,additional selectable marker genes, preferably encoding antibiotic orherbicide resistance, unique multiple cloning sites providing formultiple sites to insert DNA sequences or genes encoded in theexpression cassette and sequences that enhance transformation ofprokaryotic and eukaryotic cells.

Another vector that is useful for expression in both plant andprokaryotic cells is the binary Ti plasmid (as disclosed in Schilperoortet al., U.S. Pat. No. 4,940,838) as exemplified by vector pGA582. Thisbinary Ti plasmid vector has been previously characterized by An(Methods in Enzymology. 153:292 (1987)) and is available from Dr. An.This binary Ti vector can be replicated in prokaryotic bacteria such asE. coli and Agrobacterium. The Agrobacterium plasmid vectors can be usedto transfer the expression cassette to dicot plant cells, and undercertain conditions to monocot cells, such as rice cells. The binary Tivectors preferably include the nopaline T DNA right and left borders toprovide for efficient plant cell transformation, a selectable markergene, unique multiple cloning sites in the T border regions, the co/E1replication of origin and a wide host range replicon. The binary Tivectors carrying an expression cassette of the invention can be used totransform both prokaryotic and eukaryotic cells, but is preferably usedto transform dicot plant cells.

In Vitro Screening of Expression Cassettes:

Once the expression cassette is constructed and subcloned into asuitable plasmid, it can be screened for the ability to substantiallyinhibit the translation of an mRNA coding for a seed storage protein bystandard methods such as hybrid arrested translation. For example, forhybrid selection or arrested translation, a preselected antisense DNAsequence is subcloned into an SP6/T7 containing plasmids (as supplied byProMega Corp.). For transformation of plants cells, suitable vectorsinclude plasmids such as described herein. Typically, hybrid arresttranslation is an in vitro assay that measures the inhibition oftranslation of an mRNA encoding a particular seed storage protein. Thisscreening method can also be used to select and identify preselectedantisense DNA sequences that inhibit translation of a family orsubfamily of zein protein genes. As a control, the corresponding senseexpression cassette is introduced into plants and the phenotype assayed.

DNA Delivery of the DNA Molecules into Host Cells:

The present invention generally includes steps directed to introducing aferuloyl-CoA:monolignol transferase nucleic acids, such as a preselectedcDNA encoding the selected feruloyl-CoA:monolignol transferase enzyme,into a recipient cell to create a transformed cell. In some instances,the frequency of occurrence of cells taking up exogenous (foreign) DNAmay be low. Moreover, it is most likely that not all recipient cellsreceiving DNA segments or sequences will result in a transformed cellwherein the DNA is stably integrated into the plant genome and/orexpressed. Some may show only initial and transient gene expression.However, certain cells from virtually any dicot or monocot species maybe stably transformed, and these cells regenerated into transgenicplants, through the application of the techniques disclosed herein.

Another aspect of the invention is a plant with lignin-containingmonolignol ferulates (e.g., coniferyl ferulate), wherein the plant hasan introduced feruloyl-CoA:monolignol transferase nucleic acid. Theplant can be a monocotyledon or a dicotyledon. Another aspect of theinvention includes plant cells (e.g., embryonic cells or other celllines) that can regenerate fertile transgenic plants and/or seeds. Thecells can be derived from either monocotyledons or dicotyledons.Suitable examples of plant species include grasses, softwoods,hardwoods, wheat, rice, Arabidopsis, tobacco, maize, soybean, and thelike. In some embodiments, the plant or cell is a monocotyledon plant orcell. For example, the plant or cell can be a softwood plant or cell, ora maize plant or cell. In some embodiments, the plant or cell is adicotyledon plant or cell. For example, the plant or cell can be ahardwood plant or cell. The cell(s) may be in a suspension cell cultureor may be in an intact plant part, such as an immature embryo, or in aspecialized plant tissue, such as callus, such as Type I or Type IIcallus.

Transformation of plant cells can be conducted by any one of a number ofmethods known to those of skill in the art. Examples are: Transformationby direct DNA transfer into plant cells by electroporation (U.S. Pat.No. 5,384,253 and U.S. Pat. No. 5,472,869, Dekeyser et al., The PlantCell. 2:591-602 (1990)); direct DNA transfer to plant cells by PEGprecipitation (Hayashimoto et al., Plant Physiol. 93:857-863 (1990));direct DNA transfer to plant cells by microprojectile bombardment(McCabe et al., Bio/Technology. 6:923-926 (1988); Gordon-Kamm et al.,The Plant Cell. 2:603-618 (1990); U.S. Pat. No. 5,489,520; U.S. Pat. No.5,538,877; and U.S. Pat. No. 5,538,880) and DNA transfer to plant cellsvia infection with Agrobacterium. Methods such as microprojectilebombardment or electroporation can be carried out with “naked” DNA wherethe expression cassette may be simply carried on any E. coli-derivedplasmid cloning vector. In the case of viral vectors, it is desirablethat the system retain replication functions, but lack functions fordisease induction.

One method for dicot transformation, for example, involves infection ofplant cells with Agrobacterium tumefaciens using the leaf-disk protocol(Horsch et al., Science 227:1229-1231 (1985). Monocots such as Zea mayscan be transformed via microprojectile bombardment of embryogenic callustissue or immature embryos, or by electroporation following partialenzymatic degradation of the cell wall with a pectinase-containingenzyme (U.S. Pat. No. 5,384,253; and U.S. Pat. No. 5,472,869). Forexample, embryogenic cell lines derived from immature Zea mays embryoscan be transformed by accelerated particle treatment as described byGordon-Kamm et al. (The Plant Cell. 2:603-618 (1990)) or U.S. Pat. No.5,489,520; U.S. Pat. No. 5,538,877 and U.S. Pat. No. 5,538,880, citedabove. Excised immature embryos can also be used as the target fortransformation prior to tissue culture induction, selection andregeneration as described in U.S. application Ser. No. 08/112,245 andPCT publication WO 95/06128. Furthermore, methods for transformation ofmonocotyledonous plants utilizing Agrobacterium tumefaciens have beendescribed by Hiei et al. (European Patent 0 604 662, 1994) and Saito etal. (European Patent 0 672 752, 1995).

Methods such as microprojectile bombardment or electroporation arecarried out with “naked” DNA where the expression cassette may be simplycarried on any E. coli-derived plasmid cloning vector. In the case ofviral vectors, it is desirable that the system retain replicationfunctions, but lack functions for disease induction.

The choice of plant tissue source for transformation will depend on thenature of the host plant and the transformation protocol. Useful tissuesources include callus, suspensions culture cells, protoplasts, leafsegments, stem segments, tassels, pollen, embryos, hypocotyls, tubersegments, meristematic regions, and the like. The tissue source isselected and transformed so that it retains the ability to regeneratewhole, fertile plants following transformation, i.e., containstotipotent cells. Type I or Type II embryonic maize callus and immatureembryos are preferred Zea mays tissue sources. Similar tissues can betransformed for softwood or hardwood species. Selection of tissuesources for transformation of monocots is described in detail in U.S.application Ser. No. 08/112,245 and PCT publication WO 95/06128.

The transformation is carried out under conditions directed to the planttissue of choice. The plant cells or tissue are exposed to the DNA orRNA carrying the feruloyl-CoA:monolignol transferase nucleic acids foran effective period of time. This may range from a less than one secondpulse of electricity for electroporation to a 2-3 day co-cultivation inthe presence of plasmid-bearing Agrobacterium cells. Buffers and mediaused will also vary with the plant tissue source and transformationprotocol. Many transformation protocols employ a feeder layer ofsuspended culture cells (tobacco or Black Mexican Sweet corn, forexample) on the surface of solid media plates, separated by a sterilefilter paper disk from the plant cells or tissues being transformed.

Electroporation:

Where one wishes to introduce DNA by means of electroporation, it iscontemplated that the method of Krzyzek et al. (U.S. Pat. No. 5,384,253)may be advantageous. In this method, certain cell wall-degradingenzymes, such as pectin-degrading enzymes, are employed to render thetarget recipient cells more susceptible to transformation byelectroporation than untreated cells. Alternatively, recipient cells canbe made more susceptible to transformation, by mechanical wounding.

To effect transformation by electroporation, one may employ eitherfriable tissues such as a suspension cell cultures, or embryogeniccallus, or alternatively, one may transform immature embryos or otherorganized tissues directly. The cell walls of the preselected cells ororgans can be partially degraded by exposing them to pectin-degradingenzymes (pectinases or pectolyases) or mechanically wounding them in acontrolled manner. Such cells would then be receptive to DNA uptake byelectroporation, which may be carried out at this stage, and transformedcells then identified by a suitable selection or screening protocoldependent on the nature of the newly incorporated DNA.

Microprojectile Bombardment:

A further advantageous method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method,microparticles may be coated with DNA and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like.

It is contemplated that in some instances DNA precipitation onto metalparticles would not be necessary for DNA delivery to a recipient cellusing microprojectile bombardment. In an illustrative embodiment,non-embryogenic BMS cells were bombarded with intact cells of thebacteria E. coli or Agrobacterium tumefaciens containing plasmids witheither the β-glucoronidase or bar gene engineered for expression inmaize. Bacteria were inactivated by ethanol dehydration prior tobombardment. A low level of transient expression of the β-glucoronidasegene was observed 24-48 hours following DNA delivery. In addition,stable transformants containing the bar gene were recovered followingbombardment with either E. coli or Agrobacterium tumefaciens cells. Itis contemplated that particles may contain DNA rather than be coatedwith DNA. Hence it is proposed that particles may increase the level ofDNA delivery but are not, in and of themselves, necessary to introduceDNA into plant cells.

An advantage of microprojectile bombardment, in addition to it being aneffective means of reproducibly stably transforming monocots, is thatthe isolation of protoplasts (Christou et al., PNAS. 84:3962-3966(1987)), the formation of partially degraded cells, or thesusceptibility to Agrobacterium infection is not required. Anillustrative embodiment of a method for delivering DNA into maize cellsby acceleration is a Biolistics Particle Delivery System, which can beused to propel particles coated with DNA or cells through a screen, suchas a stainless steel or Nytex screen, onto a filter surface covered withmaize cells cultured in suspension (Gordon-Kamm et al., The Plant Cell.2:603-618 (1990)). The screen disperses the particles so that they arenot delivered to the recipient cells in large aggregates. It is believedthat a screen intervening between the projectile apparatus and the cellsto be bombarded reduces the size of projectile aggregate and maycontribute to a higher frequency of transformation, by reducing damageinflicted on the recipient cells by an aggregated projectile.

For bombardment, cells in suspension are preferably concen-trated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themacroprojectile stopping plate. If desired, one or more screens are alsopositioned between the acceleration device and the cells to bebombarded. Through the use of techniques set forth here-in one mayobtain up to 1000 or more foci of cells transiently expressing a markergene. The number of cells in a focus which express the exogenous geneproduct 48 hours post-bombardment often range from about 1 to 10 andaverage about 1 to 3.

In bombardment transformation, one may optimize the prebombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment can influence transformation frequency.Physical factors are those that involve manipulating theDNA/microprojectile precipitate or those that affect the path andvelocity of either the macro- or microprojectiles. Biological factorsinclude all steps involved in manipulation of cells before andimmediately after bombardment, the osmotic adjustment of target cells tohelp alleviate the trauma associated with bombardment, and also thenature of the transforming DNA, such as linearized DNA or intactsupercoiled plasmid DNA.

One may wish to adjust various bombardment parameters in small scalestudies to fully optimize the conditions and/or to adjust physicalparameters such as gap distance, flight distance, tissue distance, andhelium pressure. One may also minimize the trauma reduction factors(TRFs) by modifying conditions which influence the physiological stateof the recipient cells and which may therefore influence transformationand integration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation. Execution of such routineadjustments will be known to those of skill in the art.

An Example of Production and Characterization of Stable TransgenicMaize:

After effecting delivery of a feruloyl-CoA:monolignol transferasenucleic acid to recipient cells by any of the methods discussed above,the transformed cells can be identified for further culturing and plantregeneration. As mentioned above, in order to improve the ability toidentify transformants, one may desire to employ a selectable orscreenable marker gene as, or in addition to, the expressibleferuloyl-CoA:monolignol transferase nucleic acids. In this case, onewould then generally assay the potentially transformed cell populationby exposing the cells to a selective agent or agents, or one wouldscreen the cells for the desired marker gene trait.

Selection:

An exemplary embodiment of methods for identifying transformed cellsinvolves exposing the bombarded cultures to a selective agent, such as ametabolic inhibitor, an antibiotic, herbicide or the like. Cells whichhave been transformed and have stably integrated a marker geneconferring resistance to the selective agent used, will grow and dividein culture. Sensitive cells will not be amenable to further culturing.

To use the bar-bialaphos or the EPSPS-glyphosate selective system,bombarded tissue is cultured for about 0-28 days on nonselective mediumand subsequently transferred to medium containing from about 1-3 mg/lbialaphos or about 1-3 mM glyphosate, as appropriate. While ranges ofabout 1-3 mg/l bialaphos or about 1-3 mM glyphosate can be employed, itis proposed that ranges of at least about 0.1-50 mg/l bialaphos or atleast about 0.1-50 mM glyphosate will find utility in the practice ofthe invention. Tissue can be placed on any porous, inert, solid orsemi-solid support for bombardment, including but not limited to filtersand solid culture medium. Bialaphos and glyphosate are provided asexamples of agents suitable for selection of transformants, but thetechnique of this invention is not limited to them.

An example of a screenable marker trait is the red pigment producedunder the control of the R-locus in maize. This pigment may be detectedby culturing cells on a solid support containing nutrient media capableof supporting growth at this stage and selecting cells from colonies(visible aggregates of cells) that are pigmented. These cells may becultured further, either in suspension or on solid media. The R-locus isuseful for selection of transformants from bombarded immature embryos.In a similar fashion, the introduction of the C1 and B genes will resultin pigmented cells and/or tissues.

The enzyme luciferase is also useful as a screenable marker in thecontext of the present invention. In the presence of the substrateluciferin, cells expressing luciferase emit light which can be detectedon photographic or X-ray film, in a luminometer (or liquid scintillationcounter), by devices that enhance night vision, or by a highly lightsensitive video camera, such as a photon counting camera. All of theseassays are nondestructive and transformed cells may be cultured furtherfollowing identification. The photon counting camera is especiallyvaluable as it allows one to identify specific cells or groups of cellswhich are expressing luciferase and manipulate those in real time.

It is further contemplated that combinations of screenable andselectable markers may be useful for identification of transformedcells. For example, selection with a growth inhibiting compound, such asbialaphos or glyphosate at concentrations below those that cause 100%inhibition followed by screening of growing tissue for expression of ascreenable marker gene such as luciferase would allow one to recovertransformants from cell or tissue types that are not amenable toselection alone. In an illustrative embodiment embryogenic Type IIcallus of Zea mays L. can be selected with sub-lethal levels ofbialaphos. Slowly growing tissue was subsequently screened forexpression of the luciferase gene and transformants can be identified.

Regeneration and Seed Production:

Cells that survive the exposure to the selective agent, or cells thathave been scored positive in a screening assay, are cultured in mediathat supports regeneration of plants. One example of a growth regulatorthat can be used for such purposes is dicamba or 2,4-D. However, othergrowth regulators may be employed, including NAA, NAA+2,4-D or perhapseven picloram. Media improvement in these and like ways can facilitatethe growth of cells at specific developmental stages. Tissue can bemaintained on a basic media with growth regulators until sufficienttissue is available to begin plant regeneration efforts, or followingrepeated rounds of manual selection, until the morphology of the tissueis suitable for regeneration, at least two weeks, then transferred tomedia conducive to maturation of embryoids. Cultures are typicallytransferred every two weeks on this medium. Shoot development signalsthe time to transfer to medium lacking growth regulators.

The transformed cells, identified by selection or screening and culturedin an appropriate medium that supports regeneration, can then be allowedto mature into plants. Developing plantlets are transferred to soillessplant growth mix, and hardened, e.g., in an environmentally controlledchamber at about 85% relative humidity, about 600 ppm CO₂, and at about25-250 microeinsteins/sec·m² of light. Plants can be matured either in agrowth chamber or greenhouse. Plants are regenerated from about 6 weeksto 10 months after a transformant is identified, depending on theinitial tissue. During regeneration, cells are grown on solid media intissue culture vessels. Illustrative embodiments of such vessels arepetri dishes and Plant Con™. Regenerating plants can be grown at about19° C. to 28° C. After the regenerating plants have reached the stage ofshoot and root development, they may be transferred to a greenhouse forfurther growth and testing.

Mature plants are then obtained from cell lines that are known toexpress the trait. In some embodiments, the regenerated plants are selfpollinated. In addition, pollen obtained from the regenerated plants canbe crossed to seed grown plants of agronomically important inbred lines.In some cases, pollen from plants of these inbred lines is used topollinate regenerated plants. The trait is genetically characterized byevaluating the segregation of the trait in first and later generationprogeny. The heritability and expression in plants of traits selected intissue culture are of particular importance if the traits are to becommercially useful.

Regenerated plants can be repeatedly crossed to inbred plants in orderto introgress the feruloyl-CoA:monolignol transferase nucleic acids intothe genome of the inbred plants. This process is referred to asbackcross conversion. When a sufficient number of crosses to therecurrent inbred parent have been completed in order to produce aproduct of the backcross conversion process that is substantiallyisogenic with the recurrent inbred parent except for the presence of theintroduced feruloyl-CoA:monolignol transferase nucleic acids, the plantis self-pollinated at least once in order to produce a homozygousbackcross converted inbred containing the feruloyl-CoA:monolignoltransferase nucleic acids. Progeny of these plants are true breeding.

Alternatively, seed from transformed monocot plants regenerated fromtransformed tissue cultures is grown in the field and self-pollinated togenerate true breeding plants.

Seed from the fertile transgenic plants can then be evaluated for thepresence and/or expression of the feruloyl-CoA:monolignol transferasenucleic acids (or the feruloyl-CoA:monolignol transferase enzyme).Transgenic plant and/or seed tissue can be analyzed forferuloyl-CoA:monolignol transferase expression using standard methodssuch as SDS polyacrylamide gel electrophoresis, liquid chromatography(e.g., HPLC) or other means of detecting a product offeruloyl-CoA:monolignol transferase activity (e.g., coniferyl ferulate).

Once a transgenic seed expressing the feruloyl-CoA:monolignoltransferase sequence and having an increase in monolignol ferulates inthe lignin of the plant is identified, the seed can be used to developtrue breeding plants. The true breeding plants are used to develop aline of plants with an increase in the percent of monolignol ferulatesin the lignin of the plant while still maintaining other desirablefunctional agronomic traits. Adding the trait of increased monolignolferulate production in the lignin of the plant can be accomplished byback-crossing with this trait and with plants that do not exhibit thistrait and studying the pattern of inheritance in segregatinggenerations. Those plants expressing the target trait in a dominantfashion are preferably selected. Back-crossing is carried out bycrossing the original fertile transgenic plants with a plant from aninbred line exhibiting desirable functional agronomic characteristicswhile not necessarily expressing the trait of an increased percent ofmonolignol ferulates in the lignin of the plant. The resulting progenyare then crossed back to the parent that expresses the increasedmonolignol ferulate trait. The progeny from this cross will alsosegregate so that some of the progeny carry the trait and some do not.This back-crossing is repeated until an inbred line with the desirablefunctional agronomic traits, and with expression of the trait involvingan increase in monolignol ferulates (e.g., coniferyl ferulate) withinthe lignin of the plant. Such expression of the increased percentage ofmonolignol ferulates in plant lignin can be expressed in a dominantfashion.

Subsequent to back-crossing, the new transgenic plants can be evaluatedfor an increase in the weight percent of monolignol ferulatesincorporated into the lignin of the plant. This can be done, forexample, by NMR analysis of whole plant cell walls (Kim, H., and Ralph,J. Solution-state 2D NMR of ball-milled plant cell wall gels inDMSO-d₆/pyridine-d₅. (2010) Org. Biomol. Chem. 8(3), 576-591; Yelle, D.J., Ralph, J., and Frihart, C. R. Characterization of non-derivatizedplant cell walls using high-resolution solution-state NMR spectroscopy.(2008) Magn. Reson. Chem. 46(6), 508-517; Kim, H., Ralph, J., andAkiyama, T. Solution-state 2D NMR of Ball-milled Plant Cell Wall Gels inDMSO-d₆. (2008) BioEnergy Research 1(1), 56-66; Lu, F., and Ralph, J.Non-degradative dissolution and acetylation of ball-milled plant cellwalls; high-resolution solution-state NMR. (2003) Plant J. 35(4),535-544). The new transgenic plants can also be evaluated for a batteryof functional agronomic characteristics such as lodging, kernelhardness, yield, resistance to disease, resistance to insect pests,drought resistance, and/or herbicide resistance.

Plants that may be improved by these methods include but are not limitedto oil and/or starch plants (canola, potatoes, lupins, sunflower andcottonseed), forage plants (alfalfa, clover and fescue), grains (maize,wheat, barley, oats, rice, sorghum, millet and rye), grasses(switchgrass, prairie grass, wheat grass, sudangrass, sorghum,straw-producing plants), softwood, hardwood and other woody plants(e.g., those used for paper production such as poplar species, pinespecies, and eucalyptus). In some embodiments the plant is a gymnosperm.Examples of plants useful for pulp and paper production include mostpine species such as loblolly pine, Jack pine, Southern pine, Radiatapine, spruce, Douglas fir and others. Hardwoods that can be modified asdescribed herein include aspen, poplar, eucalyptus, and others. Plantsuseful for making biofuels and ethanol include corn, grasses (e.g.,miscanthus, switchgrass, and the like), as well as trees such as poplar,aspen, willow, and the like. Plants useful for generating dairy forageinclude legumes such as alfalfa, as well as forage grasses such asbromegrass, and bluestem.

Determination of Stably Transformed Plant Tissues:

To confirm the presence of the feruloyl-CoA:monolignol transferasenucleic acids in the regenerating plants, or seeds or progeny derivedfrom the regenerated plant, a variety of assays may be performed. Suchassays include, for example, molecular biological assays available tothose of skill in the art, such as Southern and Northern blotting andPCR; biochemical assays, such as detecting the presence of a proteinproduct, e.g., by immunological means (ELISAs and Western blots) or byenzymatic function; plant part assays, such as leaf, seed or rootassays; and also, by analyzing the phenotype of the whole regeneratedplant.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA may only be expressed in particular cells ortissue types and so RNA for analysis can be obtained from those tissues.PCR techniques may also be used for detection and quantification of RNAproduced from introduced feruloyl-CoA:monolignol transferase nucleicacids. PCR also be used to reverse transcribe RNA into DNA, usingenzymes such as reverse transcriptase, and then this DNA can beamplified through the use of conventional PCR techniques. Furtherinformation about the nature of the RNA product may be obtained byNorthern blotting. This technique will demonstrate the presence of anRNA species and give information about the integrity of that RNA. Thepresence or absence of an RNA species can also be determined using dotor slot blot Northern hybridizations. These techniques are modificationsof Northern blotting and also demonstrate the presence or absence of anRNA species.

While Southern blotting and PCR may be used to detect theferuloyl-CoA:monolignol transferase nucleic acid in question, they donot provide information as to whether the preselected DNA segment isbeing expressed. Expression may be evaluated by specifically identifyingthe protein products of the introduced feruloyl-CoA:monolignoltransferase nucleic acids or evaluating the phenotypic changes broughtabout by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focusing, or by chromatographictechniques such as ion exchange, liquid chromatography or gel exclusionchromatography. The unique structures of individual proteins offeropportunities for use of specific antibodies to detect their presence informats such as an ELISA assay. Combinations of approaches may beemployed with even greater specificity such as Western blotting in whichantibodies are used to locate individual gene products that have beenseparated by electrophoretic techniques. Additional techniques may beemployed to absolutely confirm the identity of theferuloyl-CoA:monolignol transferase such as evaluation by amino acidsequencing following purification. The Examples of this application alsoprovide assay procedures for detecting and quantifyingferuloyl-CoA:monolignol transferase activity. Other procedures may beadditionally used.

The expression of a gene product can also be determined by evaluatingthe phenotypic results of its expression. These assays also may takemany forms including but not limited to analyzing changes in thechemical composition, morphology, or physiological properties of theplant. Chemical composition may be altered by expression of preselectedDNA segments encoding storage proteins which change amino acidcomposition and may be detected by amino acid analysis.

DEFINITIONS

As used herein, “isolated” means a nucleic acid or polypeptide has beenremoved from its natural or native cell. Thus, the nucleic acid orpolypeptide can be physically isolated from the cell or the nucleic acidor polypeptide can be present or maintained in another cell where it isnot naturally present or synthesized.

As used herein, a “native” nucleic acid or polypeptide means a DNA, RNAor amino acid sequence or segment that has not been manipulated invitro, i.e., has not been isolated, purified, and/or amplified.

The following non-limiting Examples illustrate how aspects of theinvention have been developed and can be made and used.

Example 1 Materials and Methods

This Example illustrates some methods that can be employed to make anduse the invention.

Angelica sinensis Tissue Collection and Total RNA Extraction

One- and two-year-old field grown Angelica sinensis plants (MountainGardens Herbs), were transplanted into Readi-Earth and grown for twomonths in a greenhouse to recover. The single root of a two-year plantwas harvested, cut into small pieces, and ground in liquid nitrogen to afine powder. Total RNA was extracted by adding 100 mg of powderedAngelica sinensis root tissue to 1 ml Trizol buffer (Invitrogen) andincubating for 15 minutes while vortexing at room temperature. One-fifthvolume of chloroform was added and incubated for an additional 15minutes. After centrifugation at 15000×g for 35 minutes at 4° C., theaqueous phase was extracted with ⅕ volume of chloroform. Total RNA wasprecipitated from the aqueous phase by adding ⅕ volume of a solutioncontaining 1 M sodium chloride and 0.8 M sodium citrate and ⅕ volume ofisopropyl alcohol. The RNA was collected by centrifugation at 12,000×gand the pellet was washed in 70% ethanol, dried and dissolved inRNase-free water.

Residual DNA was removed by DNase digestion using the RNase-free DNaseKit (Qiagen), following manufacturer's guidelines. RNA quality wasassessed using an Agilent 2100 Bioanalyzer.

Library Quality cDNA Synthesis and 454 Sequencing

A cDNA library was constructed from Angelica sinensis root RNA using theCreator SMART cDNA Library Construction Kit (Clontech). First-strandcDNA was synthesized by combining 1 μg of RNA with 10 μM SMART IV Oligo,10 pM of modified CDS III/3′ cDNA synthesis primer 5′-TAG AGG CCG AGGCGG CCG ACA TGT TTT GTT TTT TTT TCT TTT TTT TTT N-3′ (SEQ ID NO:3) withPAGE purification (Integrated DNA Technologies), and deionized water toa final volume of 5 μL and incubated at 72° C. for 2 minutes. Sampleswere cooled on ice for 2 minutes and a solution of 2 μL 5× First StrandBuffer, 20 nM dithiothreitol (Creator SMART cDNA Library ConstructionKit, Clontech), 10 nM dNTP mix and 200 units SuperScript II ReverseTranscriptase (Invitrogen) was added to each reaction tube. Samples wereincubated at 42° C. for 1 hour, and then placed on ice to terminatefirst strand cDNA synthesis.

Double stranded cDNA was amplified from first strand cDNA synthesisreactions by combining 2 μL of first strand cDNA, 10 μL 10× Advantage 2PCR Buffer (Advantage 2 Polymerase Mix, Clontech), 20 nM dNTP mix(Invitrogen), 20 pM 5′ PCR Primer (Creater SMART cDNA LibraryConstruction Kit, Clontech), 20 pM Modified CDS III/3′ PCR Primer (IDT,see sequence above), 2 μL 50× Advantage 2 Polymerase Mix (Clontech), anddeionized water to a final volume of 100 μL. This reaction was placed ina thermal cycler, preheated to 95° C., and cycled 24 times (95° C. for1.25 minutes and 68° C. for 6 minutes). A 5 μL aliquot of each doublestranded cDNA reaction was analyzed by gel electrophoresis. The cDNA wassubjected to Proteinase K digestion by adding 40 μg of Proteinase K withincubation at 45° C. for 20 minutes. A solution of 50% phenol and 50%chloroform was used to extract proteins from each cDNA sample followedby two chloroform extraction. The double stranded cDNA was pooled fromall reactions and precipitated by adding 1/10 volume of 3 M sodiumacetate pH 4.8, 20 μg glycogen, and 2.5 volumes ethanol at roomtemperature. After centrifugation at 15000×g, the cDNA pellet was washedwith 80% ethanol, dried and dissolved in 79 μL deionized water. Thedouble stranded cDNA was digested with SfiI to remove concatenatedprimers and size fractionated using Chroma Spin+TE-1000 Columns(Clontech) to remove short fragments. Fractions were analyzed by agarosegel electrophoresis and the fractions with sizes above 500 base pairswere pooled. cDNA was submitted to the Genomics Core at Michigan StateUniversity for Roche 454 sequencing using the 454 GSFLX TitaniumSequencer.

Amplification and Cloning of Feruloyl-CoA:Monolignol Transferase (FMT)

cDNA was synthesized from the Angelica sinensis root total RNA, using

Superscript III Reverse Transcriptase (Invitrogen). After DNasedigestion, 5 μg of total RNA was added to 0.5 μg Oligo d(T)₁₂₋₁₈, 10 nMdNTP mix (Invitrogen) and DEPC water to a volume of 13 μL. The reactionmixture was incubated at 65° C. for 5 minutes. After cooling the sampleon ice for 2 minutes, 4 μL of 5× First-strand Buffer, 100 nM DTT, 40units RNase OUT and 200 units Superscript III Reverse Transcriptase(Invitrogen) were added and incubated at 50° C. for 60 minutes. Thereaction was inactivated by heating to 70° C. for 15 minutes and storedon ice. The FMT coding sequence was amplified using 5′-AAA AAA GCA GGCTTC ATG ACG ATC ATG GAG GTT CAA GTT-3′ (SEQ ID NO:4) and 5′-GTA CAA GAAAGC TGG GTT CTA GGA AGC GAA AGC AGA GAT-3′ (SEQ ID NO:5)oligonucleotides (Integrated DNA Technologies) as forward and reversegene specific primers with partial Gateway attB1 and attB2 attachmentsites. Using the Platinum Pfx DNA Polymerase kit (Invitrogen), 2 μL10×Pfx Amplification Buffer, 7.5 nM dNTP mix, 25 nM magnesium sulfate,10 mM of each primer, 2.5 units of Plantinum Pfx DNA Polymerase anddeionized water to a final volume of 20 μL was added to 200 ng cDNA. Thesample was denatured at 94° C. for 4 minutes, followed by 25 cycles of94° C. for 30 seconds, 55° C. for 30 seconds, and 68° C. for 1 minute 45seconds. After a cooling the sample to 4° C., a second PCR reaction wascompleted, as described above, using 5′-GGGG ACA AGT TTG TAC AAA AAA GCAGGC T-3′ (SEQ ID NO:6) and 5′-GGG AC CAC TTT GTA CAA GAA AGC TGG GT-3′(SEQ ID NO:7) oligonucleotides (Integrated DNA Technologies) as forwardand reverse primers and 2.5 μL of the first PCR reaction to add fulllength Gateway attB1 and attB2 attachment sites to the coding sequence.After amplification, the reaction was analyzed by electrophoresis on a0.8% agarose gel and the PCR product was purified using the QIAquick GelExtraction Kit (Qiagen), following manufacturer's guidelines.

The amplified FMT coding sequence was cloned into the Gateway entryvector pDONR221 (Invitrogen) using the BP Clonase II Enzyme Mix(Invitrogen). After purification, 150 ng of PCR product was added to 150ng of pDONR221 entry vector, to a final volume of 4 μL with TE buffer,and 1 μL BP Clonase II Enzyme Mix. The reaction was incubated overnightat room temperature, inactivated by adding 1 μg Proteinase K andincubating at 37° C. for 10 minutes. After cooling on ice, 2.5 μL of thereaction was used to transform One Shot Top 10 Chemically Competent E.coli Cells (Invitrogen) according to manufacturer's guidelines. Thetransformants were grown at 37° C. overnight on LB agar platescontaining and 50 μg/ml Kanamycin. Single colonies were picked and grownin LB media containing 50 μg/ml Kanamycin overnight at 37° C. PlasmidDNA was purified from these cultures using the QIAprep Spin Miniprep Kit(Qiagen), according to manufacturer's guidelines. Samples were submittedfor high throughput sequencing, using the M13 forward and M13 reverseprimers (Invitrogen) at the Michigan State University Genomics Core, andcompared to the 454 sequencing data to verify coding sequence usingDNASTAR Lasergene 8 software.

Sequences in entry vectors were inserted into pDEST17 vector using 150ng of plasmid DNA from the entry clone, 150 ng of pDEST17 vector and 1μL LR Clonase II Enzyme Mix. The reaction was incubated overnight atroom temperature. Transformation of competent cells was completed asdescribed above. Transformants were selected on LB agar platescontaining 100 μg/ml Ampicillin. Clones were screened by PCR using GotaqHot Start Green Master Mix (Promega) by adding 10 μL of the 2× mastermix to 10 mM of each gene specific primer, deionized water to finalvolume of 20 μL. This PCR reaction was denatured at 94° C. for 3 minutesthen cycled 25 times through 94° C. for 30 seconds, 55° C. for 30seconds, 72° C. for 1 minute 45 seconds, with a final elongation step at72° C. for 5 minutes before cooling to 4° C. Each reaction was analyzedby gel electrophoresis. Clones were then transformed into One Shot BL21Chemically Competent E. coli Cells (Invitrogen), according tomanufacturer's guidelines, for expression.

Expression of Feruloyl-CoA:Monolignol Transferase (FMT) in E. coli

Cultures of BL21 E. coli containing FMT nucleic acids in the expressionvector were grown at 37° C. overnight in 5 ml LB media containing 100μg/ml ampicillin. The cultures were then added to 1 L of LB mediacontaining 100 μg/ml ampicillin and grown to an OD600 of 0.4 to 0.5.Protein expression in the cells was induced by adding 1 mM of isopropylβ-D-1-thiogalactopyranoside (IPTG) and the cells were incubated for 6hours at 22° C. Cells were harvested by centrifugation at 4° C. andpellets were stored at −80° C. The pellets were suspended in 10 ml ofbinding buffer, a solution containing 20 mM Tris-hydrochloride pH 8, 0.5M sodium chloride, 1 mM 2-mercaptoethanol and cells were lysed using aFrench press. The extract was then centrifuged at 50,000×g for 30minutes at 4° C. to separate soluble and insoluble protein fractions.The soluble protein fraction in the supernatant was collected and theinsoluble protein fraction was suspended in 10 ml of suspension buffer.Both fractions were analyzed for expression on an SDS-PAGE gel.

Purification of E. coli expressed Feruloyl-CoA:Monolignol Transferase(FMT)

HIS-tagged FMT was purified using an AKTA purifier (GE Healthcare)operated with UNICORN 5.11—workstation version (GE Healthcare) and aprotocol modified from the manufacturer's guidelines. Four 5 ml HiTrapdesalting columns (GE Healthcare) were equilibrated with binding buffer.A 5 ml aliquot of the soluble protein was injected onto the desaltingcolumn and eluted with binding buffer at a flow rate of 1 ml/minute.Fractions with the highest protein concentrations, as indicated byhigher UV absorbance, were collected in 1 ml fractions. These fractionswere applied to a 1 ml H isTrap HP column (GE Healthcare), conditionedand charged with 0.1 M NiSO₄, according to manufacturer's guidelines, ata flow rate of 0.1 ml/minute. The column was washed with 5 ml of bufferA (20 mM Tris-hydrochloride pH 8, 0.5 M sodium chloride, 1 mM2-mercaptoethanol, and 20 mM imidazole) then bound protein was eluted at1 ml/minute with a 20 ml linear gradient from buffer A to buffer B (20mM Tris-hydrochloride pH 8, 0.5 M sodium chloride, 1 mM2-mercaptoethanol, and 500 mM imidazole). Fractions containing proteinwere collected and analyzed by SDS-PAGE. Fractions with the highestconcentration of FMT were combined and desalted using an Amicon Ultracel10K membrane filter (Millipore).

Feruloyl-CoA:Monolignol Transferase (FMT) Enzymatic Assay

The feruloyl-CoA, p-coumaroyl-CoA, and caffeoyl-CoA substrates used inthe FMT assay were enzymatically synthesized using the tobacco4-coumarate-CoA-ligase (4CL) with a c-terminal HIS tag in pCRT7/CT TOPO,provided by Eran Pichersky. Following a method modified from Beuerle andPichersky (Anal. Biochem. 302(2): 305-12 (2001)) 3.3 mg of ferulic acid,coumaric acid or caffeic acid, 2 mg coenzyme A, and 6.9 mg ATP wereadded to 50 mM Tris-hydrochloride pH 8 and 2.5 mM magnesium chloride ina final volume of 10 ml. The reaction was started by adding 0.25 mg 4CL,protein purified as described by the method of Beurerle and Pichershy.After a five-hour incubation at room temperature, additional 6.9 mg ATP,2 mg coenzyme A, and 0.25 mg purified 4CL were added and the reactionwas incubated overnight. The CoA esters were purified on an SPEcartridge as described in Beuerle and Pichersky (2001).

The FMT activity assay contained 100 mM MOPS pH 6.8, 1 mM dithiothreitol(DTT), 1 mM feruloyl-CoA, 1 mM coniferyl alcohol, 3.9 μg of purified FMTprotein and deionized water to a volume of 50 μL. After a 30-minuteincubation, 1 μL of 10 M hydrochloric acid was added to stop thereaction. Because the product synthesized in the reaction, coniferylferulate (CAFA), is insoluble, 50 μL of methanol was added to solubilizethe CAFA. Prior to UPLC, protein and insoluble material were removed byfiltering through an Amicon Ultracel 10K membrane filter (Millipore).The flow-through was analyzed using an Acquity Ultra Performance LC withan Acquity UPLC BEH C18 1.7 μm 2.1×100 mm column and the Acquity Consoleand Empower 2 Software, all from Waters Corporation. The solvents usedin this method were solvent A, 0.1% trifluoroacetic acid, and solvent B,100% acetonitrile. Samples were analyzed using the following gradientconditions, 13% B, for 5 minutes, 1 minute linear gradient to 42% B,held for 4 minutes, 1 minute linear gradient to 100% B, held for 1minutes and 3 minutes at 13% B with a flow rate of 0.3 ml/minute. Thismethod was then used to analyze a 10 μL injection of each assayreaction; standards for each of the substrates along with chemicallysynthesized CAFA were used to determine retention times for eachcompound.

Size Exclusion Chromatography of FMT

A 100 μL sample of protein purified by immobilized metal ion affinitychromatography (IMAC) was loaded onto a Superdex 75 10/300 GL gelfiltration column (GE Healthcare), equilibrated with 100 mM MOPS pH 6.8.The protein was eluted with the same buffer at a constant flow rate of0.1 ml/minute and collected in 0.5 ml fractions. Aliquots of the proteinsample prior to gel filtration, and each of the fractions near theelution peak were analyzed for protein content by SDS-PAGE gelelectrophoresis. Protein containing fractions were analyzed to determinethe amount of FMT activity, as described above.

NMR

To confirm the identification based on the chromatogram peakcomparisons, the reaction product, which was insoluble before additionof methanol, was centrifuged to pellet the coniferyl ferulate, which wasdissolved in perdeuteroacetone and analyzed by NMR. The proton NMRspectrum, FIG. 3A, unambiguously confirmed the authenticity of theconiferyl ferulate product, particularly when compared with the spectrumfrom the independently synthesized coniferyl ferulate (described below).For absolute confirmation, ¹³C NMR data was also obtained via a 2D¹H-¹³C correlation (HSQC) spectrum (for the protonated carbons, FIG. 3B)and a 2D ¹H-¹³C long-range correlation (HMBC) spectrum (not shown, butdata for all carbons is given on FIG. 3B).

Synthesis of Authentic Coniferyl Ferulate

The synthesis was similar to that described for the related compound,coniferyl p-coumarate (Lu, F., and Ralph, J. Facile synthesis of4-hydroxycinnamyl p-coumarates. (1998) J. Agr. Food Chem. 46(8),2911-2913). Thus, as shown in FIG. 9, 4-acetoxyferuloyl chloride wasprepared from ferulic acid by acetylation followed by chlorination usingSOCl₂ according to a previous method (Helm, R. F., Ralph, J., andHatfield, R. D. Synthesis of feruloylated and p-coumaroylated methylglycosides. (1992) Carbohydr. Res. 229(1), 183-194).

4-Acetoxyconiferaldaldehyde was prepared in 94-96% yield by acetylationof coniferaldehyde with acetic anhydride/pyridine and then reduced withborane/tert-butylamine complex to give the corresponding alcohol, asfollows. The 4-acetoxyconiferaldehyde was dissolved in methylenechloride to which borane/tert-butylamine complex (1.5 equiv) was added.The mixture was stirred at room temperature for 2 h, when TLC showedthat the starting material had disappeared completely. The solvent wasevaporated at 40° C. under reduced pressure. The residue was hydrolyzedwith 0.5 M H₂SO₄ in ethanol/water (1:1) for 1.5 h. Most of the ethanolwas removed by evaporation, and the product was extracted with ethylacetate. The ethyl acetate solution was washed with saturated NH₄Cl anddried over MgSO₄. Evaporation of the ethyl acetate gave the product,4-acetoxyconiferyl alcohol as a pale yellow oil (96% yield); ¹H NMR(acetone-d₆) δ 2.31 (3H, s, OAc), 3.83 (3H, s, OAc), 3.90 (1H, t, J) 5.5Hz, γ-OH), 4.22 (2H, dt, J) 5.5, 1.7 Hz, γ), 6.38 (1H, dt, J) 15.9, 5.2Hz, β), 6.58 (1H, dt, J=15.9, 1.7 Hz, α), 6.97 (2H, m, A5/6), 7.15 (s,1H, A2); ¹³C NMR δ 20.5 (OAc), 56.2 (OMe), 63.1 (γ), 110.9 (A2), 119.5(A6), 123.6 (A5), 129.3 (a), 131.4 (β), 137.2 (A1), 140.2 (A4), 152.3(A3), 169.0 (OAc).

4-Acetoxyconiferylferulate.

Coupling of 4-acetoxyferuloyloyl chloride with 4-acetoxyconiferylalcohol was efficiently carried out using 4-(dimethylamino)-pyridine(DMAP). Thus, 4-acetoxyconiferyl alcohol and 4-acetoxyferuloyl chloridewere dissolved in dry CH₂Cl₂ (120 mL) to which DMAP (0.25 equiv) andEt₃N (0.85 equiv) were added. The mixture was stirred for 2 h, when TLC[CHCl₃/EtOAc (5:1)] showed the starting material was converted into afaster moving compound. The solution was diluted with CH₂Cl₂ and washedsuccessively with aqueous 3% HCl and saturated NH₄Cl. Drying over MgSO₄,evaporation, and purification by flash chromatography [CHCl₃/EtOAc(19:1)] gave the diacetate of coniferyl ferulate (94%) as a pale yellowoil.

Coniferyl Ferulate.

The above diacetate (0.195 mmol) was dissolved in pyrrolidine (1 mL).Once dissolution was complete, the pyrrolidine solution was diluted with50 mL of ethyl acetate and washed with 1 M H₂SO₄ (3×20 mL) and saturatedNH₄Cl (2×20 mL). After drying over MgSO₄ and evaporation, the resultingsyrup was submitted to solid phase extraction [CHCl₃/EtOAc (19:1)] toafford coniferyl ferulate (93%) as a white solid. NMR spectra are thesame as those for the FMT-enzyme generated product, as shown in FIG. 3.

Example 2 Identification and Cloning of a Feruloyl-CoA:monolignolTransferase

Mature A. sinensis plants were purchased from Mountains, Gardens andHerbs (North Carolina) and RNA was extracted from the roots of theseplants. This RNA was used to synthesize double-stranded cDNA. The cDNAwas sequenced using a Roche GSFLX Titanium Sequencer and 736,017sequences were obtained. The sequences were assembled into 62425 contigsusing CAP3 (Huang, X., A contig assembly program based on sensitivedetection of fragment overlaps. (1992) Genomics 14: 18-25). Theconsensus sequence for each contig was searched against all proteinsfrom Arabidopsis and the NCBI non-redundant protein databases using theBLASTX software program (Altschul S, Gish W, Miller W, Myers E, LipmanD. Basic local alignment search tool. (1990) J Mol Biol 215(3),403-410). The sequences were sorted by abundance and filtered to showonly sequences annotated as being within a “transferase family,” whichis the annotation in the TAIR9 database assigned to members of the BAHDclass of acyltransferases.

Two very abundant BAHD acyltransferases were identified as well as anumber of such enzymes with lower EST counts. These two sequences werecloned by PCR from an A. sinensis cDNA pool using oligonucleotidesdesigned to amplify their coding regions. The coding region of the A.sinensis sequences was transferred to the expression vector pDEST17using Gateway technology. This vector adds an amino-terminal 6×HIS-tagto the protein, which allows for affinity purification by immobilizedmetal affinity chromatography (IMAC). E. coli clones containing therecombinant protein where grown and induced to produce recombinantprotein. The enzyme was purified from the E. coli protein extract usingIMAC.

Purified recombinant enzyme was assayed for FMT activity using areaction mixture containing 2 mM coniferyl alcohol, 0.5 feruloyl-CoA,100 mM HEPES pH 7.4 and 1 mM DTT. The second most abundant BAHDacyltransferase gene when incubated with Coniferyl alcohol andferuloyl-CoA produced a compound with the retention time of authenticconiferyl ferulate (CAFA) (FIG. 2). The product produced was mostlyinsoluble in water. The addition of methanol to 50% after stopping theenzyme with acid was required to analyze the product by UPLC. Theinsolubility of the product made partial purification easy as theproduct was separated from the substrates by centrifugation.

This partial purified product was analyzed by NMR. The identity of theproduct as CAFA was confirmed by ¹H-NMR (FIG. 3). The enzyme was testedwith p-coumaryl alcohol (FIG. 4) and sinapyl alcohol (FIG. 5) inaddition to coniferyl alcohol (FIG. 2). The enzyme is active with allthree monolignols, i.e., p-coumaryl alcohol, coniferyl alcohol andsinapyl alcohol. The enzyme was tested with p-coumaroyl-CoA (FIG. 6) andcaffeoyl-CoA (FIG. 7) as well as feruloyl-CoA (FIG. 2). The enzyme has astrong preference for feruloyl-CoA as can be seen by comparison of FIGS.2, 6 and 7. In FIGS. 6 and 7, very little product is produced fromp-coumaroyl-CoA and caffeoyl-CoA substrates. However, substantialproduct is formed when feruloyl-CoA is used instead (FIG. 2).

The IMAC purified FMT had a few lower molecular weight proteins as shownin FIG. 8. These lower molecular proteins are likely proteolyticfragments of FMT as determined by analysis of tryptic digests of thesebands by mass spectrometry. To ensure that the major band wasresponsible for the activity, FMT was further purified usingsize-exclusion chromatography. The FMT activity elutes coincident withthe major protein band (FIG. 8).

Example 3 Analysis of Transgenic Poplar Containing the FMT Sequence

This Example illustrates the expression and enzymatic activity observedin poplar trees that were genetically modified to express the Angelicasinensis feruloyl-CoA:monolignol transferase nucleic acids describedherein.

Methods

Hybrid poplar (Populus alba×grandidentata) was transformed usingAgrobacterium tumefaciens EHA105 employing a common leaf diskinoculation. Two constructs were created to drive the expression of FMTin poplar: 1) 35S::YFP-FMT (cauliflower mosaic virus ubiquitous 35Spromoter with an N-terminal tagged Yellow Fluorescent Protein), and 2)CesA8::YFP-FMT (poplar xylem-specific secondary cell wall specificcellulose synthase 8 promoter with an N-terminal tagged YellowFluorescent Protein). The binary plasmids were inserted into EHA105using the freeze-thaw technique, and incubated overnight in liquid WoodyPlant Media (WPM) supplemented with 100 μM acetosyringone. Leaf diskswere cut and co-cultured with EHA105 for one hour at room temperature,blotted dry and plated abaxailly onto WPM supplemented with 0.1 μM eachα-naphthalene acetic acid (NAA), 6-benzylaminopurine (BA), andthiadiazuron (TDZ) and solidified with 3% (w/v) agar and 1.1% (w/v)phytagel (WPM 0.1/0.1/0.1). After three days the discs were transferredto WPM 0.1/0.1/0.1 supplemented with carbenicillin disodium (500 mg L⁻¹)and cefotaxime sodium salt (250 mg L⁻¹). Following three additionaldays, the discs were transferred to WPM 0.1/0.1/0.1 containingcarbenicillin, cefotaxime and hygromycin (25 mg L⁻¹). After five weeks,shoots and callus material were transferred to WPM with agar andphytagel, 0.01 μM BA, carbenicillin, cefotaxime and hygromycin. Onceindividual shoots were visible, plantlets were transferred to solidifiedWPM with 0.01 μM NAA and carbenicillin, cefotaxime and hygromycin toinduce rooting. After two consecutive five-week periods on this media,shoot tips were isolated to solidified antibiotic-free WPM with 0.01 μMNAA.

Plants were confirmed as transgenic by PCR screening of genomic DNAemploying gene specific oligonucleotides. All shoot cultures, includingtransgenic and non-transformed wild-type lines, were maintained on solidWPM with 0.01 μM NAA in GA-7 vessels at 22° C. under a 16-hourphotoperiod with an average photon flux of 50 mmol m⁻² s⁻¹ untilout-planting to the greenhouse. Plants were then transferred to soil andgrown under supplemental lights (at about 300 W m²) on flood tables andwatered with fertigated water daily in a greenhouse.

Purification of YFP-FMT was via GFPtrap_A (Chromotek) following themanufactures guidelines. Briefly, leaves from transgenic 1-year poplartrees were ground to a powder in liquid nitrogen and 250 mg powder ofeach ground leaf sample was separately suspended in 300 μl 100 mM sodiumphosphate pH 6. An aliquot of 5 ul was added to the FMT enzyme assaydescribed in the foregoing Examples. After 45 minutes of incubation, thereaction was stopped with 100 mM hydrochloric acid, and the productswere solubilized with the addition of methanol to a concentration of50%. The protein and insoluble materials were removed by filtrationthrough an Amicon Ultracel 10K membrane filter (Millipore). Controlreactions were also completed using a protein extract from wild typehybrid poplar, as well as the standard no enzyme control. These sampleswere analyzed by western blot and the UPLC method described in theExamples above. Formation of coniferyl ferulate was also detected bycomparison of the UPLC traces of leaf extracts with authentic coniferylferulate.

Results

As shown in FIG. 10, FMT activity was identified in extracts fromtransgenic poplar lines containing the Angelica sinensis FMT byobserving a product peak at the same retention time as the authenticstandard (FIG. 10B). No such peak was observed for wild type popularleaf extracts or in the no enzyme control. Similarly, FMT proteinexpression was detected by western blot analysis only in leaves frompoplar trees that had been genetically modified to express the Angelicasinensis FMT (FIG. 10A).

Example 4 Transgenic Arabidopsis with the Angelica sinensis FMT

This Example illustrates that other plant species can readily betransformed with the Angelica sinensis feruloyl-CoA:monolignoltransferase nucleic acids described herein to express an enzymaticallyactive FMT.

Methods:

Arabidopsis were transformed by standard procedures with the Angelicasinensis feruloyl-CoA:monolignol transferase nucleic acids describedherein. As a control some samples of Arabidopsis were transformed withan empty vector that did not contain the Angelica sinensis FMT. FMTexpression was detected by Reverse Transcriptase PCR of protein isolatedfrom the transgenic Arabidopsis leaves. Enzymatic activity by theexpressed FMT was detected using the assay described in Example 1.

Results

As illustrated in FIG. 11, the transgenic Arabidopsis plants express anenzymatically active Angelica sinensis feruloyl-CoA:monolignoltransferase. FIG. 11A shows the products of Reverse Transcriptase PCRamplification of transcripts from Arabidopsis leaves transformed withempty vector or with a vector expressing the FMT transcript. As shown,FMT transcripts were detected only when reverse transcriptase was added(+RT) to the PCR reaction mixture, and not when reverse transcriptasewas absent (−RT) from the PCR reaction mixture. A PCR product of theexpected size for the FMT enzyme (1326 base pairs) was visible only inthe reaction containing total RNA from Arabidopsis transformed with theAngelica sinensis FMT when the reverse transcriptase is present.

FIG. 11B shows representative UPLC traces illustrating FMT activity inground stems from Arabidopsis transformed with the FMT from Angelicasinensis (see, bottom panel). The absorbance for each of the substrates,coniferyl alcohol (1) and feruloyl-CoA (2) and for the product,coniferyl ferulate (3), was detected at 280 nm (solid line) and at 340nm (dotted line). The top panel of FIG. 11B shows the results of controlreactions of stems transformed with empty vector (top panel). Coniferylferulate (3) is detected only when protein from the transformedArabidopsis-FMT stems was added.

These data indicate that plants can readily be transformed with theAngelica sinensis nucleic acids described herein and such transformedplants can readily express an enzymatically activeferuloyl-CoA:monolignol transferase that incorporates monolignolferulates such as coniferyl ferulate into plant tissues.

Example 5 Isolation of Hibiscus cannabinus (Kenaf) FMT

This Example illustrates isolation of the Hibiscus cannabinus (Kenaf)feruloyl-CoA:monolignol transferase nucleic acids and expression of anenzymatically active FMT.

Materials and Methods

Hibiscus cannabinus (Kenaf) stem sections were collected and stored inRNAlater (Qiagen) until processing. The tissue was then removed from theRNAlater solution and ground to a powder in liquid nitrogen. Total RNAwas extracted by adding 100 mg of powdered Hibiscus cannabinus stemsections to 1 ml Trizol buffer (Invitrogen) and incubating for 15minutes while vortexing at room temperature. One-fifth volume ofchloroform was added and the mixture was incubated for an additional 15minutes. After centrifugation at 15000×g for 35 minutes at 4° C., theaqueous phase was extracted with ⅕ volume of chloroform. Total RNA wasprecipitated from the aqueous phase by adding ⅕ volume of a solutioncontaining 1 M sodium chloride and 0.8 M sodium citrate and ⅕ volumes ofisopropyl alcohol. The RNA was collected by centrifugation at 12,000×gand the pellet was washed in 70% ethanol, dried and dissolved inRNase-free water. Residual DNA was removed by DNase digestion using theRNase-free DNase Kit (Qiagen), following manufacturer's guidelines. RNAquality was assessed using an Agilent 2100 Bioanalyzer. Total RNA fromHibiscus cannabinus was submitted to the Genomics Core at Michigan StateUniversity for Roche 454 sequencing using the 454 GSFLX TitaniumSequencer.

Candidate Selection

Ferulate monolignol transferase (FMT) candidates were chosen from theKenaf_CLC 454 sequencing database by searching for “transferase familyproteins” that have no close homologs in Arabidopsis thaliana. Thecandidates with the largest number of EST sequences were amplified andcloned.

Cloning of Hibiscus cannabinus FMT

cDNA was synthesized from the Hibiscus cannabinus stem sections totalRNA, using Superscript III Reverse Transcriptase (Invitrogen). AfterDNase digestion, 5 μg of total RNA was added to 0.5 μg Oligo d(T)₁₂₋₁₈,10 nM dNTP mix (Invitrogen) and DEPC water to a volume of 13 μL. Thereaction mixture was incubated at 65° C. for 5 minutes. After coolingthe sample on ice for 2 minutes, 4 μL of 5× First-strand Buffer, 100 nMDTT, 40 units RNase OUT and 200 units Superscript III ReverseTranscriptase (Invitrogen) were added and incubated at 50° C. for 60minutes. The reaction was inactivated by heating to 70° C. for 15minutes and stored on ice. The Hibiscus cannabinus FMT coding sequencewas amplified using 5′-AAAAAAGCAGGCTTCATGGCAACCCACAGCACTATCAT-3′ (SEQ IDNO:10 and 5′-GTACAAGAAAGCTGGGTTCTAGATCACTAGAGCATCGCCGG-3′ (SEQ ID NO:11)oligonucleotides (Integrated DNA Technologies) as forward and reversegene specific primers with partial Gateway attB1 and attB2 attachmentsites. Using the Platinum Pfx DNA Polymerase kit (Invitrogen), 2 μL 10×Pfx Amplification Buffer, 7.5 nM dNTP mix, 25 nM magnesium sulfate, 10mM of each primer, 2.5 units of Plantinum Pfx DNA Polymerase anddeionized water to a final volume of 20 μL was added to 200 ng cDNA. Thesample was denatured at 94° C. for 4 minutes, followed by 25 cycles of94° C. for 30 seconds, 52° C. for 30 seconds, and 68° C. for 2 minutes.After a cooling the sample to 4° C., a second PCR reaction wascompleted, as described above with a 55° C. annealing temperature, using5′-GGGG ACA AGT TTG TAC AAA AAA GCA GGC T-3′ (SEQ ID NO:12) and 5′-GGGAC CAC TTT GTA CAA GAA AGC TGG GT-3′ (SEQ ID NO:13) oligonucleotides(Integrated DNA Technologies) as forward and reverse primers and 2.5 μLof the first PCR reaction to add full length Gateway attB1 and attB2attachment sites to the coding sequence. After amplification, thereaction was analyzed by electrophoresis on a 0.8% agarose gel and thePCR product was purified using the QIAquick Gel Extraction Kit (Qiagen),following manufacturer's guidelines.

The amplified FMT coding sequence was cloned into the Gateway entryvector pDONR221 (Invitrogen) using the BP Clonase II Enzyme Mix(Invitrogen). After purification, 150 ng of PCR product was added to 150ng of pDONR221 entry vector, to a final volume of 4 μL with Tris-EDTA(TE) buffer, and 1 μL BP Clonase II Enzyme Mix. The reaction wasincubated overnight at room temperature, inactivated by adding 1 μgProteinase K and incubating at 37° C. for 10 minutes. After cooling onice, 2.5 μL of the reaction was used to transform One Shot Top 10Chemically Competent E. coli Cells (Invitrogen) according tomanufacturer's guidelines. The transformants were grown at 37° C.overnight on LB agar plates containing and 50 μg/ml Kanamycin. Singlecolonies were picked and grown in LB media containing 50 μg/ml Kanamycinovernight at 37° C. Plasmid DNA was purified from these cultures usingthe QIAprep Spin Miniprep Kit (Qiagen), according to manufacturer'sguidelines. Samples were submitted for high throughput sequencing, usingthe M13 forward and M13 reverse primers (Invitrogen), along with5′-CGCACTCGGTTTGTGATGGC-3′ (SEQ ID NO:14) and5′-TTCACAGCTTTCGAGAGCGGTC-3′ (SEQ ID NO:15) as two gene specificprimers, at the Michigan State University Genomics Core. This sequencedata was compared to the 454 sequencing data to verify coding sequenceusing DNASTAR Lasergene 8 Sequence Manager software.

The following were the Hibiscus cannabinus (Kenaf) nucleotide andprotein sequences chosen for expression. Nucleotide sequence SEQ IDNO:8:

1 ATGGCAACCC ACAGCACTAT CATGTTCTCA GTCGATAGAA 41ACGATGTCGT GTTTGTCAAA CCCTTCAAAC CTACACCCTC 81ACAGGTTCTA TCTCTCTCCA CCATCGACAA TGATCCCAAC 121CTTGAGATCA TGTGCCATAC TGTTTTTGTG TATCAAGCCA 161ATGCCGATTT CGATGTTAAG CCCAAGGATC CAGCTTCCAT 201AATCCAGGAA GCACTCTCCA AGCTCTTGGT TTATTACTAT 241CCCTTAGCGG GGAAGATGAA GAGGGAGACC GATGGAAAAC 281TTCGAATCGC TTGCACTGCC GACGATAGCG TGCCCTTCTT 321AGTAGCCACC GCCGATTGCA AGCTCTCGTC GTTGAACCAC 361TTGGATGGCA TAGATGTTCA TACCGGGAAA GAATTCGCCT 401TGGATTTTGC ATCCGAATCC GACGGTGGCT ATTATCACCC 441TCTGGTCATG CAGGTGACGA AGTTCATATG CGGAGGGTTC 481ACCATCGCTT TGAGTTTATC GCACTCGGTT TGTGATGGCT 521TCGGTGCAGC TCAGATCTTT CAAGCATTGA CCGAGCTCGC 561AAGTGGCAGG AACGAGCCCT CGGTTAAACC CGTGTGGGAG 601AGGCAACTAT TAGTGGCGAA ACCGGCCGAG GAAATCCCTC 641GGTCGATTGT CGATAAGGAC TTGTCGGCAG CTTCACCGTA 681TCTGCCGACA ACCGACATAG TCCATGCCTG CTTTTATGTA 721ACCGAGGAGA GTATAAAAAC ACTGAAAATG AATCTGATCA 761AAGAAAGCAA AGATGAGAGT ATAACCAGTC TCGAGGTCCT 801TTCAGCCTAT ATATGGAGAG CAAGGTTTAG AGCATTGAAA 841TTGAGTCCAG ATAAAACCAC AATGCTCGGC ATGGCCGTAG 881GCATACGACG CACCGTGAAA CCACGGTTGC CCGAAGGATA 921CTACGGGAAT GCTTTCACCT CGGCAAATAC GGCCATGACC 961GGGAAGGAAC TCGACCAAGG ACCGCTCTCG AAAGCTGTGA 1001AACAAATCAA GGAGAGCAAA AAGCTTGCTT CGGAGAATGA 1041CTATATCTGG AACTTGATGA GCATTAACGA GAAGCTGAGA 1081GAACTGAATT CGAAGTTCGA AGCGGCCGCC GGTTCAACCA 1121TGGTCATAAC AGATTGGAGG CGGTTGGGAC TATTGGAAGA 1161TGTGGATTTT GGATGGAAAG GTAGCGTAAA CATGATACCA 1201CTGCCGTGGA ACATGTTCGG GTACGTGGAT TTGGTTCTTT 1241TATTGCCTCC TTGTAAACTG GACCAATCGA TGAAAGGCGG 1281TGCTAGAGTG TTGGTTTCCT TTCCCACGGC TGCTATTGCC 1321AAATTCAAGG AAGAAATGGA TGCTCTCAAA CATGATAACA 1361AGGTTGCCGG CGATGCTCTA GTGATCTAGThe SEQ ID NO:8 nucleic acid encodes a Hibiscus cannabinus (Kenaf)feruloyl-CoA:monolignol transferase enzyme with the following amino acidsequence (SEQ ID NO:9).

1 MATHSTIMFS VDRNDVVFVK PFKPTPSQVL SLSTIDNDPN 41LEIMCHTVFV YQANADFDVK PKDPASIIQE ALSKLLVYYY 81PLAGKMKRET DGKLRIACTA DDSVPFLVAT ADCKLSSLNH 121LDGIDVHTGK EFALDFASES DGGYYHPLVM QVTKFICGGF 161TIALSLSHSV CDGFGAAQIF QALTELASGR NEPSVKPVWE 201RQLLVAKPAE EIPRSIVDKD LSAASPYLPT TDIVHACFYV 241TEESIKTLKM NLIKESKDES ITSLEVLSAY IWRARFRALK 281LSPDKTTMLG MAVGIRRTVK PRLPEGYYGN AFTSANTAMT 321GKELDQGPLS KAVKQIKESK KLASENDYIW NLMSINEKLR 361ELNSKFEAAA GSTMVITDWR RLGLLEDVDF GWKGSVNMIP 401LPWNMFGYVD LVLLLPPCKL DQSMKGGARV LVSFPTAAIA 441 KFKEEMDALK HDNKVAGDAL VI

Sequences in entry vectors were inserted into pDEST17 vector using 150ng of plasmid DNA from the Kenaf FMT entry clone, 150 ng of pDEST17vector and 1 μL LR Clonase II Enzyme Mix. The reaction was incubatedovernight at room temperature. Transformation of competent cells wascompleted as described above. Transformants were selected on LB agarplates containing 100 μg/ml Ampicillin. Clones were screened by PCRusing Gotaq Hot Start Green Master Mix (Promega) by adding 10 μL of the2× master mix to 10 mM of each gene specific primer with partial GatewayattB1 and attB2 attachment sites as described above, deionized water tofinal volume of 20 μL. This PCR reaction was denatured at 94° C. for 3minutes then cycled 25 times through 94° C. for 30 seconds, 52° C. for30 seconds, 72° C. for 2 minutes, with a final elongation step at 72° C.for 5 minutes before cooling to 4° C. Each reaction was analyzed by gelelectrophoresis. Clones were then transformed into One Shot BL21Chemically Competent E. coli Cells (Invitrogen), according tomanufacturer's guidelines, for expression.

Expression of FMT in E. coli

Cultures of BL21 E. coli containing the Kenaf FMT in the expressionvector were grown at 37° C. overnight in 5 ml LB media containing 100μg/ml ampicillin, then added to 500 ml of LB media containing 100 μg/mlampicillin and grown to an OD600 of 0.3 to 0.4. The culture was theninduced by adding 1 mM of Isopropyl β-D-1-thiogalactopyranoside, IPTG,and incubated overnight at 18° C. Cells were harvested by centrifugationat 4° C. and pellets were stored at −80° C. The pellets were suspendedin 10 ml of binding buffer, a solution containing 20 mMTris-hydrochloride pH 8, 0.5 M sodium chloride, 1 mM 2-mercaptoethanoland cells were lysed using a French press. The extract was thencentrifuged at 50,000×g for 30 minutes at 4° C. to separate soluble andinsoluble protein fractions. The soluble protein fraction, supernatant,was collected and the insoluble protein fraction was suspended in 10 mlof suspension buffer. Both fractions were analyzed for expression on anSDS-PAGE gel.

Purification of E. coli Expressed FMT

HIS-tagged Kenaf FMT was purified using an AKTA purifier (GE Healthcare)operated with UNICORN 5.11—workstation version (GE Healthcare) and aprotocol modified from the manufacturer's guidelines. Four 5 ml HiTrapDesalting columns (GE Healthcare) were equilibrated with binding buffer.A 5 ml aliquot of the soluble protein was injected onto the desaltingcolumn and eluted with binding buffer at a flow rate of 1 ml/minute.Fractions with the highest protein concentrations, as indicated byhigher UV absorbance, were collected in 1 ml fractions. These fractionswere applied to a 1 ml H isTrap HP column (GE Healthcare), conditionedand charged with 0.1 M NiSO₄ according to manufacturer's guidelines, ata flow rate of 0.1 ml/minute. The column was washed with 5 ml of bufferA (20 mM Tris-hydrochloride pH 8, 0.5 M sodium chloride, 1 mM2-mercaptoethanol, and 20 mM imidazole) then bound protein was eluted at1 ml/minute with a 20 ml linear gradient from buffer A to buffer B (20mM Tris-hydrochloride pH 8, 0.5 M sodium chloride, 1 mM2-mercaptoethanol, and 500 mM imidazole). Fractions containing proteinwere collected and analyzed by SDS-PAGE. Fractions with the highestconcentration of Kenaf FMT were combined and desalted using an AmiconUltracel 10K membrane filter (Millipore).

FMT Enzymatic Assay

The feruloyl CoA, p-coumaroyl CoA, and caffeoyl CoA substrates used inthe FMT assay were enzymatically synthesized using the tobacco4-coumarate CoA-ligase (4CL) with a c-terminal HIS tag in pCRT7/CT TOPO.Following a method modified from Beuerle and Pichersky (2001) 3.3 mg offerulic acid, coumaric acid or caffeic acid, 2 mg coenzyme A, and 6.9 mgATP were 50 mM Tris-hydrochloride pH 8, 2.5 mM magnesium chloride in afinal volume of 10 ml. The reaction was started by adding 0.25 mg 4CLprotein, purified as described by the method of Beurerle and Pichershy.After a five-hour incubation at room temperature, an additional 6.9 mgATP, 2 mg coenzyme A, and 0.25 mg purified 4CL were added and thereaction was incubated overnight. The CoA esters were purified on an SPEcartridge as described in Beuerle and Pichersky (2001).

The FMT activity assay contained 100 mM sodium phosphate buffer pH 6, 1mM dithiothreitol (DTT), 1 mM feruloyl CoA, 1 mM coniferyl alcohol, 0.5μg of purified Kenaf FMT protein and deionized water to a volume of 50μL. After a 45-minute incubation, 100 mM hydrochloric acid was added tostop the reaction. Because the product synthesized in the reaction,coniferyl ferulate (CAFA), is partially insoluble, 50 μL of methanol wasadded to solubilize the CAFA. Prior to UPLC, protein and insolublematerial were removed by filtering through an Amicon Ultracel 10Kmembrane filter (Millipore). The flow-through was analyzed using anAcquity Ultra Performance LC with an Acquity UPLC BEH C18 1.7 μm 2.1×100mm column and the Acquity Console and Empower 2 Software, all fromWaters Corporation. The solvents used in this method were solvent A,0.1% trifluoroacetic acid, and solvent B, 100% acetonitrile. Sampleswere analyzed using the following gradient conditions, 13% B, for 5minutes, 1 minute linear gradient to 42% B, held for 4 minutes, 1 minutelinear gradient to 100% B, held for 1 minute and 3 minutes at 13% B witha flow rate of 0.3 ml/minute. This method was then used to analyze a 10μL injection of each assay reaction; standards for each of thesubstrates along with chemically synthesized CAFA were used to determineretention times for each compound.

FIGS. 12A and 12B illustrate the expression, purification and enzymeactivity for FMT from Hibiscus cannabinus. FIG. 12A shows that theHibiscus cannabinus FMT is expressed in E. coli BL21 cells. The Hibiscuscannabinus FMT was expressed with an N-terminal 6×His tag in the pDEST17 vector (Invitrogen) and the soluble protein (˜50 kDa) was purifiedover a Ni²⁺ column using an AKTA purifier (GE Healthcare).

Fractions 29 and 30 from the Ni²⁺ column that contained purified proteinwere assayed for FMT activity. FIG. 12B shows the products of an FMTenzyme assay of fractions 29 and 30 after UPLC separation. The productsof the FMT enzyme assay were detected by absorbance at 280 nm (solidline) and 340 nm (dotted line) for the substrates coniferyl alcohol (1)and feruloyl-CoA (2). A control reaction with no enzyme is shown at thetop of FIG. 12B. The products of the assay containing the Hibiscuscannabinus FMT enzyme are shown in the bottom panel of FIG. 12B. Theproduction of coniferyl ferulate (3) is visible only when the Hibiscuscannabinus FMT enzyme was present in the assay (bottom panel). Theproduct and substrate peaks were identified by comparison to syntheticstandards.

FIG. 13 shows an alignment of the Hibiscus cannabinus and Angelicasinensis feruloyl-CoA:monolignol transferase sequences. As illustrated,the Hibiscus cannabinus and Angelica sinensis feruloyl-CoA:monolignoltransferases share only about 23% sequence identity. When similar aminoacid substitutions are considered, the Hibiscus cannabinus and Angelicasinensis feruloyl-CoA:monolignol transferases share only about 41%sequence similarity.

All patents and publications referenced or mentioned herein areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each such referenced patent or publicationis hereby specifically incorporated by reference to the same extent asif it had been incorporated by reference in its entirety individually orset forth herein in its entirety. Applicants reserve the right tophysically incorporate into this specification any and all materials andinformation from any such cited patents or publications.

The specific methods and compositions described herein arerepresentative of preferred embodiments and are exemplary and notintended as limitations on the scope of the invention. Other objects,aspects, and embodiments will occur to those skilled in the art uponconsideration of this specification, and are encompassed within thespirit of the invention as defined by the scope of the claims. It willbe readily apparent to one skilled in the art that varying substitutionsand modifications may be made to the invention disclosed herein withoutdeparting from the scope and spirit of the invention. The inventionillustratively described herein suitably may be practiced in the absenceof any element or elements, or limitation or limitations, which is notspecifically disclosed herein as essential. The methods and processesillustratively described herein suitably may be practiced in differingorders of steps, and the methods and processes are not necessarilyrestricted to the orders of steps indicated herein or in the claims. Asused herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, a reference to “a nucleic acid” or “apolypeptide” includes a plurality of such nucleic acids or polypeptides(for example, a solution of nucleic acids or polypeptides or a series ofnucleic acid or polypeptide preparations), and so forth. Under nocircumstances may the patent be interpreted to be limited to thespecific examples or embodiments or methods specifically disclosedherein. Under no circumstances may the patent be interpreted to belimited by any statement made by any Examiner or any other official oremployee of the Patent and Trademark Office unless such statement isspecifically and without qualification or reservation expressly adoptedin a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intent in the use ofsuch terms and expressions to exclude any equivalent of the featuresshown and described or portions thereof, but it is recognized thatvarious modifications are possible within the scope of the invention asclaimed. Thus, it will be understood that although the present inventionhas been specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims and statements of theinvention.

The following statements of the invention are intended to summarize someaspects of the invention according to the foregoing description given inthe specification.

STATEMENTS OF THE INVENTION

-   1. An isolated nucleic acid encoding a feruloyl-CoA:monolignol    transferase wherein the nucleic acid can selectively hybridize to a    DNA with a SEQ ID NO:8 sequence.-   2. The isolated nucleic acid of statement 1, wherein the nucleic    acid selectively hybridizes to a DNA with a SEQ ID NO:8 sequence    under stringent hybridization conditions.-   3. The isolated nucleic acid of statement 2, wherein the stringent    hybridization conditions comprise a wash in 0.1×SSC, 0.1% SDS at 65°    C.-   4. The isolated nucleic acid of any of statements 1-3, wherein the    nucleic acid that selectively hybridizes to a DNA with a SEQ ID NO:8    sequence has at least about 70% sequence identity with SEQ ID NO:8.-   5. The isolated nucleic acid of any of statements 1-4, wherein the    nucleic acid encodes a feruloyl-CoA:monolignol transferase that can    catalyze the synthesis of monolignol ferulate(s) from monolignol(s)    and feruloyl-CoA.-   6. The isolated nucleic acid of statement 5, wherein the monolignol    is coniferyl alcohol, p-coumaryl alcohol, sinapyl alcohol or a    combination thereof.-   7. The isolated nucleic acid of any of statements 1-6, wherein the    nucleic acid encodes a Hibiscus cannabinus (Kenaf)    feruloyl-CoA:monolignol transferase polypeptide with a SEQ ID NO:9    or SEQ ID NO:16 sequence.-   8. The isolated nucleic acid of any of statements 1-7, wherein the    nucleic acid encodes a feruloyl-CoA:monolignol transferase that can    catalyze the synthesis of monolignol ferulate(s) from a    monolignol(s) and feruloyl-CoA with at least about 50% of the    activity of a feruloyl-CoA:monolignol transferase with the SEQ ID    NO:9 or SEQ ID NO:16.-   9. A transgenic plant cell comprising the isolated nucleic acid of    any of statements 1-8.-   10. A transgenic plant comprising the plant cell of statement 9 or    the isolated nucleic acid of any of statements 1-8.-   11. An expression cassette comprising the feruloyl-CoA:monolignol    transferase nucleic acid of any of statements 1-8 operably linked to    a promoter functional in a host cell.-   12. The expression cassette of statement 11, which further comprises    a selectable marker gene.-   13. The expression cassette of statement 11 or 12, further    comprising plasmid DNA.-   14. The expression cassette of any of statements 11-13, wherein the    expression cassette is within an expression vector.-   15. The expression cassette of any of statements 11-14, wherein the    promoter is a promoter functional during plant development or    growth.-   16. The expression cassette of any of statements 11-15, wherein the    promoter is a poplar xylem-specific secondary cell wall specific    cellulose synthase 8 promoter, cauliflower mosaic virus promoter,    Z10 promoter from a gene encoding a 10 kD zein protein, Z27 promoter    from a gene encoding a 27 kD zein protein, pea rbcS gene or actin    promoter from rice.-   17. A plant cell comprising the expression cassette of any of    statements 11-16.-   18. The plant cell of statement 17, wherein the plant cell is a    monocot cell.-   19. The plant cell of statement 17, wherein the plant cell is a    maize, grass or softwood cell.-   20. The plant cell of statement 17, wherein the plant cell is a    dicot cell.-   21. The plant cell of statement 17, wherein the plant cell is a    hardwood cell.-   22. A plant comprising the expression cassette of any of statements    11-16.-   23. The plant of statement 22, wherein the plant is a monocot.-   24. The plant of statement 22, wherein the plant is a grass, maize    or softwood.-   25. The plant of statement 22, wherein the plant is a gymnosperm.-   26. The plant of statement 22, wherein the plant is a dicot.-   27. The plant of statement 22, wherein the dicot is a hardwood.-   28. A method for incorporating monolignol ferulates into lignin of a    plant, comprising:    -   a) stably transforming plant cells with the expression cassette        of any of statements 11-16 to generate transformed plant cells;    -   b) regenerating the transformed plant cells into at least one        transgenic plant, wherein feruloyl-CoA:monolignol transferase is        expressed in at least one transgenic plant in an amount        sufficient to incorporate monolignol ferulates into the lignin        of the transgenic plant.-   29. The method of statement 28, wherein the transgenic plant is    fertile.-   30. The method of statement 28 or 29, further comprising recovering    transgenic seeds from the transgenic plant, wherein the transgenic    seeds comprise the nucleic acid encoding a feruloyl-CoA:monolignol    transferase.-   31. The method of any of statements 28-30, wherein the plant is a    monocot.-   32. The method of any of statements 28-31, wherein the plant is a    grass, maize or softwood plant.-   33. The method of any of statements 28-32, wherein the plant is a    gymnosperm.-   34. The method of statement 28, wherein the plant is a dicot.-   35. The method of statement 34, wherein the dicot plant is a    hardwood.-   36. The method of any of statements 28-35, wherein the lignin in the    plant comprises at least 1% monolignol ferulate.-   37. The method of any of statements 28-36, wherein the lignin in the    plant comprises at least 5% monolignol ferulate.-   38. The method of any of statements 28-37, wherein the lignin in the    plant comprises at least 10% monolignol ferulate.-   39. The method of any of statements 28-38, wherein the lignin in the    plant comprises at least 20% monolignol ferulate.-   40. The method of any of statements 28-39, wherein the lignin in the    plant comprises at least 25% monolignol ferulate.-   41. The method of any of statements 28-40, wherein the lignin in the    plant comprises about 1-30% monolignol ferulate, or about 2-30%    monolignol ferulate.-   42. The method of any of statements 28-41, further comprising    breeding a fertile transgenic plant to yield a progeny plant that    has an increase in the percentage of monolignol ferulates in the    lignin of the progeny plant relative to the corresponding    untransformed plant.-   43. The method of any of statements 28-42, further comprising    breeding a fertile transgenic plant to yield a progeny plant that    has an increase in the percentage of monolignol ferulates in the    lignin of the progeny plant as a dominant trait while still    maintaining functional agronomic characteristics relative to the    corresponding untransformed plant.-   44. The method of any of statements 28-43, wherein the transformed    plant cell is transformed by a method selected from the group    consisting of electroporation, microinjection, microprojectile    bombardment, and liposomal encapsulation.-   45. The method of any of statements 28-44, further comprising stably    transforming the plant cell with at least one selectable marker    gene.-   46. A fertile transgenic plant having an increased percent of    monolignol ferulates in the plant's lignin, the genome of which is    stably transformed by the nucleic acid of any of statements 1-8,    wherein the nucleic acid is operably linked to a promoter functional    in a host cell, and wherein the feruloyl-CoA:monolignol transferase    nucleic acid is transmitted through a complete normal sexual cycle    of the transgenic plant to the next generation.-   47. The plant of statement 46, wherein the plant is a monocot.-   48. The plant of statement 46, wherein the plant is a grass, maize    or softwood.-   49. The plant of statement 46, wherein the plant is a gymnosperm.-   50. The plant of statement 46, wherein the plant is a dicot.-   51. The plant of statement 46, wherein the percent of monolignol    ferulates in the plant's lignin is increased relative to the    corresponding untransformed plant.-   52. The plant of any of statements 46-51, wherein the percent of    monolignol ferulates in the plant's lignin is increased by at least    1% relative to the corresponding untransformed plant.-   53. The plant of any of statements 46-52, wherein the percent of    monolignol ferulates in the plant's lignin is increased by at least    2-5% relative to the corresponding untransformed plant.-   54. The plant of any of statements 46-53, wherein the lignin in the    plant comprises at least 1% monolignol ferulates.-   55. The plant of any of statements 46-54, wherein the lignin in the    plant comprises at least 5% monolignol ferulates.-   56. The plant of any of statements 46-55, wherein the lignin in the    plant comprises at least 10% monolignol ferulates.-   57. The plant of any of statements 46-56, wherein the lignin in the    plant comprises at least 20% monolignol ferulates.-   58. The plant of any of statements 46-57, wherein the lignin in the    plant comprises at least 25% monolignol ferulates.-   59. The plant of any of statements 46-58, wherein the lignin in the    plant comprises about 1-30% monolignol ferulates.-   60. A lignin isolated from a transgenic plant comprising the    isolated nucleic of any of statements 1-8.-   61. A method of making a product from a transgenic plant comprising:    -   (a) providing or obtaining a transgenic plant that includes an        isolated nucleic acid encoding a feruloyl-CoA:monolignol        transferase comprising the isolated nucleic of any of statements        1-8; and    -   (b) processing the transgenic plant's tissues under conditions        sufficient to digest to the lignin; and thereby generate the        product from the transgenic plant,    -   wherein the transgenic plant's tissues comprise lignin having an        increased percent of monolignol ferulates relative to a        corresponding untransformed plant.-   62. The method of statement 61, wherein the conditions sufficient to    digest to the lignin comprise conditions sufficient to cleave ester    bonds within monolignol ferulate-containing lignin.-   63. The method of statement 61 or 62, wherein the conditions    sufficient to digest to the lignin comprise mildly alkaline    conditions.-   64. The method of any of statements 61-63, wherein the conditions    sufficient to digest to the lignin comprise contacting the    transgenic plant's tissues with ammonia for a time and a temperature    sufficient to cleave ester bonds within monolignol    ferulate-containing lignin.-   65. The method of any of statements 61-64, wherein the conditions    sufficient to digest to the lignin would not cleave substantially    any of the ether and carbon-carbon bonds in lignin from a    corresponding plant that does not contain the isolated nucleic acid    encoding the feruloyl-CoA:monolignol transferase.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

Other embodiments are within the following claims.

1. An isolated Hibiscus cannabinus (Kenaf) feruloyl-CoA:monolignoltransferase nucleic acid with at least about 95% sequence identity tonucleic acid sequence SEQ ID NO:8.
 2. The isolated nucleic acid of claim1, wherein the nucleic acid encodes a Hibiscus cannabinus (Kenaf)feruloyl-CoA:monolignol transferase polypeptide with amino acid sequenceSEQ ID NO:9 or SEQ ID NO:16.
 3. An expression cassette comprising aferuloyl-CoA:monolignol transferase nucleic acid segment with at leastabout 95% sequence identity to nucleic acid sequence SEQ ID NO:8, and apromoter operably linked to the nucleic acid segment, wherein thepromoter is functional in a host cell.
 4. The expression cassette ofclaim 3, wherein the nucleic acid segment encodes a Hibiscus cannabinus(Kenaf) feruloyl-CoA:monolignol transferase polypeptide with amino acidsequence SEQ ID NO:9 or SEQ ID NO:16.
 5. The expression cassette ofclaim 3, wherein the promoter is a promoter functional or active duringplant development or growth.
 6. The expression cassette of claim 3,wherein the promoter is a promoter functional or active in woody tissuesof a plant.
 7. An isolated cell comprising the isolatedferuloyl-CoA:monolignol transferase nucleic acid of claim
 1. 8. Theisolated cell of claim 7, which is a microorganism or a plant cell.
 9. Aplant comprising the isolated cell of claim
 7. 10. A plant comprising aheterologous feruloyl-CoA:monolignol transferase nucleic acid segmentwith at least about 95% sequence identity to nucleic acid sequence SEQID NO:8, and a promoter operably linked to the nucleic acid segment,wherein the promoter is functional in a cell of the plant.
 11. The plantof claim 10, wherein the plant has an increased percent of monolignolferulates in the plant's lignin.
 12. The plant of claim 10, wherein thegenome of the plant is stably transformed by the isolatedferuloyl-CoA:monolignol transferase nucleic acid.
 13. The plant of claim10, wherein the feruloyl-CoA:monolignol transferase nucleic acid istransmitted through a complete normal sexual cycle of the transgenicplant to the next generation.
 14. A plant seed comprising the expressioncassette of claim
 3. 15. A plant seed comprising a heterologousferuloyl-CoA:monolignol transferase nucleic acid segment with at leastabout 95% sequence identity to nucleic acid sequence SEQ ID NO:8, and apromoter operably linked to the nucleic acid segment, wherein thepromoter is functional in a cell of the plant.
 16. A method forincreasing the content of monolignol ferulates in lignin within a plant,comprising: (a) planting the plant seed of claim 15; and (b) cultivatinga plant germinated from the plant seed, to thereby increase the contentof monolignol ferulates in the lignin within the plant.
 17. A method forincorporating monolignol ferulates into lignin of a plant, comprising:(a) stably transforming plant cells with the expression cassette ofclaim 3, to generate transformed plant cells; (b) regenerating thetransformed plant cells into at least one transgenic plant, whereinferuloyl-CoA:monolignol transferase is expressed in at least onetransgenic plant in an amount sufficient to incorporate monolignolferulates into the lignin of the transgenic plant.
 18. A fertiletransgenic plant having an increased percent of monolignol ferulates inthe plant's lignin, the genome of which is stably transformed by theexpression cassette of claim 3, wherein the feruloyl-CoA:monolignoltransferase nucleic acid is transmitted through a complete normal sexualcycle of the transgenic plant to the next generation.
 19. An isolatedcell comprising the expression cassette claim 3.